The emergence of life on Earth

Progress in Biophysics & Molecular Biology 75 (2001) 75–120

Review

The emergence of life on Earth Noam Lahava,*, Shlomo Nira, Avshalom C. Elitzurb a

Department of Soil and Water Sciences, The Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel b Bar-Ilan University, Israel

Abstract Combined top-down and bottom-up research strategies and the principle of biological continuity were employed in an attempt to reconstruct a comprehensive origin of life theory, which is an extension of the coevolution theory (Lahav and Nir, Origins of Life Evol. Biosphere (1997) 27, 377–395). The resulting theory of emergence of templated-information and functionality (ETIF) addresses the emergence of living entities from inanimate matter, and that of the central mechanisms of their further evolution. It proposes the emergence of short organic catalysts (peptides and proto-ribozymes) and feedback-loop systems, plus their template-and-sequence-directed (TSD) reactions, encompassing catalyzed replication and translation of populations of molecules organized as chemical-informational feedback loop entities, in a fluctuating (wetting–drying) environment, functioning as simplified extant molecular-biological systems. The feedback loops with their TSD systems are chemically and functionally continuous with extant living organisms and their emergence in an inanimate environment may be defined as the beginning of life. The ETIF theory considers the emergence of bio-homochirality, a primordial genetic code, information and the incorporation of primordial metabolic cycles and compartmentation into the emerging living entities. This theory helps to establish a novel measure of biological information, which focuses on its physical effects rather than on the structure of the message, and makes it possible to estimate the time needed for the transition from the inanimate state to the closure of the first feedback-loop systems. Moreover, it forms the basis for novel laboratory experiments and computer modeling, encompassing catalytic activity of short peptides and proto-RNAs and the emergence of bio-homochirality and feedback-loop systems. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Bio-homochirality; Biological information; Catalytic peptide; Genetic code’s emergence; Feedback loop Abbreviations: ETIF}emergence of templated-information and functionality ; LUCA}last universal common ancestor; mRNA}messenger ribonucleic acid; RCC}reductive citrate cycle; tRNA}transfer ribonucleic acid; TSD}template-and-sequence directed.

*Corresponding author. Fax: +972-8-947-5181. E-mail address: [email protected] (N. Lahav). 0079-6107/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 7 9 - 6 1 0 7 ( 0 1 ) 0 0 0 0 3 - 7

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N. Lahav et al. / Progress in Biophysics & Molecular Biology 75 (2001) 75–120 Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2.

Requirements of a theory of the emergence of life . . . . . . . . . . . . . . . . . . . . . . .

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3.

The theory of emergence of templated-information and functionality (ETIF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Biological (biochemical) continuity . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Central molecules: peptides, RNAs, and their building blocks . . . . . . . . . . . . . 3.3. Specific requirements of the ETIF theory . . . . . . . . . . . . . . . . . . . . . . . 3.4. Centrality of short organic catalysts . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Merging of top-down and bottom-up strategies: small peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Supporting evidence from other research areas . . . . . . . . . . . . . . . . 3.4.3. Specificity and selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.4. Bio-homochirality takeover . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Emergence of the genetic code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1. Merging of top-down and bottom-up strategies: the emergence of the primordial genetic code . . . . . . . . . . . . . . . . . 3.6. Populations of compartmentalized feedback loops and their TSD systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.1. Influx and efflux through membranes . . . . . . . . . . . . . . . . . . . . . 3.6.2. Horizontal gene transfer and a reticulated phylogenetic tree . . . . . . . . . 3.6.3. Size of vesicles and their molecular populations . . . . . . . . . . . . . . . . 3.7. Mechanism for the incorporation of novel attributes into functioning feedback-loop systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.1. Late initiations of novel feedback loops . . . . . . . . . . . . . . . . . . . . 3.8. Magnitudes of discrete evolutionary steps . . . . . . . . . . . . . . . . . . . . . . . 3.9. Biological information and its emergence: basic thermodynamic principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.1. Information and energy: physical affinities . . . . . . . . . . . . . . . . . . . 3.9.2. Information and entropy: mathematical affinities . . . . . . . . . . . . . . . 3.9.3. Information and energy: biological affinities . . . . . . . . . . . . . . . . . . 3.9.4. Information and the emergence of the first feedback loop . . . . . . . . . . . 3.9.5. Structural information content of the first feedback loops . . . . . . . . . . . 3.9.6. Biological information is everywhere . . . . . . . . . . . . . . . . . . . . . . 3.10. Primeval reticulated phylogenetic trees and the origin of life . . . . . . . . . . . . . 3.11. On the evolution of self-sustained systems . . . . . . . . . . . . . . . . . . . . . . 3.12. Fundamental attributes of primordial living entities . . . . . . . . . . . . . . . . . 3.12.1. Teleonomy and functionality . . . . . . . . . . . . . . . . . . . . . . . . 3.12.2. Evolutionary order of appearance . . . . . . . . . . . . . . . . . . . . . . 3.13. Comparison with other approaches . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.1. Simplification: replicators . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.2. Simplification: double origin . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.3. Replicators and units of selection . . . . . . . . . . . . . . . . . . . . . . 3.13.4. Comprehensive versus partial theories . . . . . . . . . . . . . . . . . . . . 3.14. The fundamental unit of life and life’s definition . . . . . . . . . . . . . . . . . . . 3.14.1. A broad characterization of life . . . . . . . . . . . . . . . . . . . . . . . 3.14.2. Historical-mechanistic definitions according to the ETIF theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3.15.

3.16.

4.

The emergence of life and its definition are model dependent . . . . . . . . . . . . . . . . . . . Time window and duration of life’s emergence . . . . . . . . 3.15.1. Synthesis and decomposition . . . . . . . . . . . . 3.15.2. The WHEN and DURATION problems . . . . . . 3.15.3. Estimates of duration of life emergence according to ETIF theory . . . . . . . . . . . . . . . . . . . . . Implications . . . . . . . . . . . . . . . . . . . . . . . . . . 3.16.1. Novel methodologies for the study of short peptide catalysts . . . . . . . . . . . . . . . . . . . . . . . 3.16.2. Catalytic properties of short RNA molecules . . . . 3.16.3. Emergence of bio-homochirality . . . . . . . . . . . 3.16.4. Emergence of a feedback loop in a test tube . . . .

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Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

1. Introduction After almost 50 years of modern research, there is no paradigm of the origin of life. Moreover, we lack a comprehensive theory addressing the entire evolutionary range of the emergence of our phylogenetic tree, starting from the synthesis of the building blocks of biochemistry through the transition from inanimate to animate matter, to the last universal common ancestor (LUCA) and ending in extant living forms. At the same time it is generally accepted that the scientific approach is capable of providing a plausible scenario for the emergence of life (see, for instance, Deamer and Fleischaker, 1994; Lahav, 1999; Morowitz, 1992; Ochman et al., 2000; W.achtersh.auser, 1988). In the theory presented below we focus first on the emergence of living entities in an inanimate environment and then briefly address the continuation of this evolutionary pathway to the LUCA. This early evolutionary era is characterized by extensive horizontal transfer of genetic material and therefore it seems that rather than a phylogenetic tree the presumed genetical continuity of this era is better represented by a phylogenetic network (also referred to as reticulated tree, or net, see Doolittle, 1999). Extending the principle of biological continuity to encompass the entire history of life, we propose that the organizational principles laid down for the emergence of the first attributes of life are also applicable for the emergence of the central attributes of life during the entire process of the emergence of our primeval phylogenetic network, followed by our phylogenetic tree. In what follows we present first the requirements from a theory of the origin of life and then present our theory of the emergence of the first living entities from inanimate matter. The theory encompasses the emergence of the most fundamental attributes of life, including feedback

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loops and template-and-sequence-directed (TSD) reactions, bio-homochirality, and biological information.

2. Requirements of a theory of the emergence of life Apparently, a discussion on the origin of life should start with a rigorous definition of life. However, this is a tough and perhaps even an impossible task. And thus, such a discussion should better be postponed until the essence of the theory under consideration is presented (see Section 3.14). A comprehensive emergence-of-life theory should encompass the basic attributes and manifestations of life, the characterization of the environment(s) in which the central attributes of the primordial living entities could have emerged, the fundamental assumptions related to these emergence processes, the era(s) in the history of Planet Earth during which life emerged from inanimate matter, and the postulated chemical and physicochemical mechanisms of the suggested scenario of this process. We assume continuity in the evolutionary establishment of the central attributes of life during the transition from inanimate matter to the realm of biology. This transition is followed by the next stages of the same continuous evolutionary process, and includes the emergence of the LUCA of our phylogenetic tree. The two relevant research methodologies are the top-down and bottom-up strategies (see Lahav, 1999; Morowitz, 1992). The former strategy searches for clues for ancient stages of life in the biological record of extant life. The latter strategy searches for clues of primordial stages of life in the geological record, including laboratory studies, model building and computer modeling. The two strategies are not independent of each other, since top-down considerations always guide the bottom-up research programs. Merging of these two oppositely directed research strategies into a unified stage in a given scenario is an essential requirement of the reconstruction of the origin of life. These considerations should thus mold the pre-requisites of any scenario addressing the transitions from inanimate to animate matter, the LUCA and our phylogenetic tree. Such a scenario encompasses the emergence and evolution of the central attributes and functions of living entities, i.e., information and information transfer, genetic code, bio-homochirality, compartmentation and metabolism, as well as those of the population of entities constituting the LUCA (Schwabe, 1985; Woese, 1998). The general requirements of a theory addressing the emergence of terrestrial life is a mechanism for the transition from inanimate to animate matter. This encompasses the first functions fulfilled by the central molecules, the geochemical characteristics of the primordial inorganic state, including its main niches and their dynamics, availability of reactants (with some specifications regarding the chemical nature of the central compounds), availability of energy sources, net increase of the number, size, and complexity of the central molecules of the process under study with time, and damaging environmental factors. The theory should also provide estimates for the time interval between a primordial state of an inorganic system and the time of emergence of the first living entities, as well as the time intervals between the first living entities and more advanced stages of evolution, such as the LUCA. Leslie Orgel once noted that the explanation of the origin of life might be much simpler than we are used to think. However, to many a layman and some scientists, the origin of life seems to abide

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beyond the realm of the known physical laws (see for instance, Pattee, 1995). It has been suggested recently that the coevolution theory (Lahav and Nir, 1997; Nir and Lahav, 1997) provides the above general requirements for the emergence of the first living entities in an inanimate world. The implications of this theory are that the emergence of life from inanimate matter (and the evolutionary transition from the first living entities towards the LUCA), neither calls for new physical laws, nor is it beyond our intellectual capability. The goal of the present work is to expand the coevolution theory so as to include not only the appearance of the first feedback loops, but also the emergence of fundamental attributes of life, such as bio-homochirality, information, genetic code and functionality, which were involved in the evolutionary pathway leading from the primordial living entities to the LUCA. This expanded coevolution theory is named the theory of the emergence of templated-information and functionality (ETIF).

3. The theory of emergence of templated-information and functionality (ETIF) The present work is an attempt to construct a general theory of the origin of life, based on the coevolution theory (Lahav and Nir, 1997; Nir and Lahav, 1997), with its terrestrial origin of life scenario. The coevolution theory serves as a biogeochemical model for the evolution of TSD syntheses of organic templates (proto-RNAs) and catalysts (peptides as well as catalytic protoRNAs, which may function also as proto-ribozymes). A fluctuating environment characterized by hydrating (cool) and dehydrating (hot) phases with cycles of consecutive organic reactions, as well as a constant supply of building blocks is assumed. Hence, we consider the existence of low concentrations of amino acids and proto-nucleotides, the building blocks of the catalysts (short peptides as well as short proto-RNAs functioning as proto-ribozymes) and of the organic templates (short proto-RNAs). The scenario starts with the catalyzed formation of primordial populations of small random peptides and proto-RNAs, based on the relatively inefficient mineral catalysts. The processes taking place are proto-RNA chain elongation and replication, loading of amino acids by prototRNAs, and the emergence of more and more efficient organic catalysts. All these processes, which are catalyzed by short peptides and proto-ribozymes, are initially very slow and inefficient, and in particular the loading reaction, since it takes relatively long time to form a population of proto-tRNA molecules (Nir and Lahav, 1997). They are gradually accelerated, however, due to an increase in the concentrations of proto-RNA oligomers and peptides, as well as the evolution of more and more efficient catalysts and templates. The resulting catalytic peptides and protoribozymes initiate a catalytic takeover process, during which the catalytic functions are gradually taken over by peptides and proto-ribozymes. During the emergence of TSD systems the fractions of TSD peptides and proto-RNA constituents rise from almost insignificance to dominance in a TSD reaction takeover process. Consequently, a phase of exponential growth in the production of peptide catalysts is reached. In parallel, an increase in the efficiency of TSD production and development is achieved by means of random compartmentation. Thus, only compartments which include the essential molecules without a large excess of other molecules would develop efficiently, whereas the other ones, lacking an essential component of the feedback loops, would

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die out. As early compartments we envisage minerals such as clays, and later on}lipid vesicles, which are more efficient compartments. The TSD system is characterized by autocatalysis, positive feedback loops and a primordial genetic code. The feedback loops carry out two fundamental functions in the form of catalyzed reactions, namely, replication and translation. These TSD reactions and their functions include coded information, bio-homochirality and metabolic cycles, where the sequence of building blocks of the template is its structural information. Other aspects of both information and function are discussed below. The theory combines (i) bottom-up and (ii) top-down considerations: its origin-of-life proposition is essentially a bottom-up approach guided by top-down extrapolation from extant biology regarding the central molecules, their interactions and functions during and after the transition from inanimate molecules to animate assemblies of molecules. These involve, for instance, geochemical considerations for the bottom-up strategy and sequencing data of the central biomolecules of extant organisms, for the top-down approach. Eventually, merging of bottom-up and top-down research strategies is expected, as discussed below. The present theory thus presents a coherent and continuous scenario, supported by many observations and a few plausible assumptions and suggestions, thus serving as a starting point for a novel experimental program. Furthermore, according to the ETIF theory each new feature in a living system is the result of the establishment of either new or modified feedback loops in the production of catalysts and templates, as well as the outcome of their reactions. It is suggested that the synthesis of feedback-loop systems has been a common denominator for all living entities during the entire history of life. The experimental methodology implied in the ETIF theory for the study of the transition from inanimate to animate assemblies of organic molecules includes laboratory experiments and computer modeling. It should culminate, however, in an attempt to synthesize the simplest living entities, i.e., feedback-loop systems, in a test-tube (see below). Our theory is essentially a simplification of extant molecular biology. We maintain that in spite of its complexity, the theory is intellectually tenable, because the principle of biological continuity, the simplification of biochemical and molecular–biological processes and the system approach involved in the theoretical considerations discussed below, are all based on features of biology that are understood reasonably well today. Hence, there is no mental barrier in comprehending the simplified model system, which describes the origin of life. Nor does this theory contrast the Second Law of thermodynamics (Elitzur, 1994). Moreover, in spite of the apparent complexity of the proposed system, it is suggested to be close to the simplest possible network of interactions needed for the emergence of the earliest forms of life. Thus, the choice of the ETIF theory for the description of the emergence of life reflects an implied fundamental assumption, namely, that the relationships between the major constituents of extant cells, i.e., proteins, nucleic acids and metabolic cycles, as embedded in molecular biology and biochemistry, can be traced, at least partially, to the emergence of life from inanimate matter. Moreover, our computer model suggests the plausibility of initiation and establishment of feedback loops under a range of molecular properties and environmental conditions, namely, a considerable robustness of the emergence of life processes. In the present work we propose a comprehensive, mechanistic scenario of the emergence of living entities constituting the primeval phylogenetic reticulated tree, which preceded our extant

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phylogenetic tree. The theory starts with the transition from inanimate to animate matter and proceeds with the establishment of the first living entities capable of evolving into the LUCA. The genetic relations among these entities constitute a primeval phylogenetic network, which is essentially a continuous, though tortuous, evolutionary pattern of pathways leading to the very beginning of our phylogenetic tree. 3.1. Biological (biochemical) continuity An important aspect of the transition from inanimate to animate matter according to the ETIF theory is a primordial arsenal of mineral catalysts (see also Tessis et al., 1999) involved in the very first reactions of adsorption and catalysis. The role of catalytic minerals was later replaced by organic catalysts during a takeover process}first by non-templated organic catalysts and templates, and then by templated organic catalysts. Thus, the biochemical continuity starts from the latter molecules and their functions, and is considered a genetic continuity. Expanding the scope of the ETIF theory to include also the involvement of, for instance, sulfides, shifts the compositional continuity considerations to the inclusion of sulfides. This possibility will not be discussed in detail in the present work. The continuity considerations can be applied only for that part of the scenario which follows the establishment of the first feedback loops and their TSD reactions. The physico-chemical mechanism of this continuity is structural information transfer by means of templates, encompassing the entire range of life’s history, between emergence of the first feedback loop and extant organisms. Like its predecessor}the coevolution theory}the ETIF theory starts with a direct back extrapolation from extant life towards the stage of transition from inanimate to animate matter, by applying the principle of biological continuity with regard to both chemical composition, reactions and functions. As such, its beginning is a bottom-up reconstruction, guided by top-down back extrapolation and simplification of extant cell biology. However, the fundamental molecular mechanisms of the ETIF theory proceed beyond the first steps of transition from inanimate to animate matter, and cover the entire evolutionary pathway that reaches the LUCA and our phylogenetic tree. The presumed biological continuity along this pathway may be exemplified by the central role of proto-tRNA: according to the ETIF theory, primordial populations of proto-tRNAs are continuous with extant tRNAs. The involvement of a population of proto-tRNAs in the peptide synthesis machines of a population of TSD systems corroborates also with the polyphyletic nature of tRNAs, according to recent interpretation of sequencing data (see Di Giulio (1999) and references therein). We shall return later to the relations between the bottom-up and top-down outlooks. 3.2. Central molecules: peptides, RNAs, and their building blocks The central molecules of the theory of ETIF (Table 1) are rather similar to those of the coevolution theory. Both the coevolution and the ETIF theories focus on one kind of organic catalyst, i.e., (i) peptides, and also consider the possibility of (ii) proto-RNAs (see Knight and Landweber, 2000; Maurel and Decout, 1999), and (iii) unspecified cofactors. In addition, metal

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Table 1 List of prerequisites of the ETIF theory 1.

Initial geochemical environment A fluctuating (wetting/cool-drying/hot) environment.

2.

Main primordial constituents (i) Amino acids, predominantly glycine and alaninea (ii) Proto-nucleotidesb (iii) Inorganic constituents such as pyrophosphate (proto-ATP), metal ions such as Mg, Fe, Mn and various adsorbing minerals (iv) Compartmentation means, i.e., amphiphilic molecules capable of forming vesicles, and/or adsorbing minerals Main primordial syntheses and reactions of non-directed oligomersc

3.

(i) Establishment of a population of short peptides (ii) Establishment of a population of short proto-RNAs 4.

Primordial compartmentation of the central oligomers (i) Adsorption onto mineral surfaces (ii) Encapsulation of the above molecules and oligomers by vesicles made of amphiphilic molecules a

The building blocks of peptides, including homochiral and catalytic molecules. The building blocks of a population of proto-RNA oligomers. c Environmental conditions which favor the availability of primordial catalysts, such as adsorbing minerals, short homochiral peptides and short proto-RNAs, as well as activating agents, conductive to condensation of activated building blocks. b

such as Fe, Mg and Mn are also implied. The dominance of peptides in extant life suggests a central role of these molecules in the emergence of life. However, the central role of RNA molecules in protein synthesis (Ban et al., 2000; Cech, 2000; Muth et al., 2000; Nissen et al., 2000) suggests a very early involvement of RNAs, and presumably also proto-RNA, in primordial translation reactions, perhaps even during the establishment of the first feedback loops with their TSD systems. For simplicity, and because so little is known presently about the exact role of peptides and RNAs in the transition from inanimate to animate, we designate the organic catalysts of the ETIF theory as peptides. When more information is gained about primordial catalysts it would be possible to ascribe specific catalytic functions to each of these molecules, namely, peptides and RNAs. It should be noted that the choice of peptides as primordial organic catalysts seems not only justified at the present stage of our understanding of the origin of life (de Duve, 1991; Lahav, 1991; Lahav and Nir, 1997), but is also crucial in practically all the stages of life’s emergence. Two important examples are synthesis of homochiral, glycine-rich catalytic peptides on the one hand and selectivity of peptides in catalytic reactions on the other, as discussed below. The ETIF theory does not specify the mechanism by which the building blocks of the first oligomers and additional central molecules are formed. Similarly, it does not address the mechanism of formation of the first metabolic cycles, such as those suggested by Lazcano and

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Miller (1999). Rather, the ETIF theory assumes that the building blocks of the primordial oligomers, which are central to the first forms of life, were readily formed in certain environmental niches of the primordial Earth. The theory focuses on the emergence of the first living entities, followed by the more evolutionary-advanced forms of life, based on these building blocks and their ‘‘manipulations’’ by fluctuating (wet–dry) environments. For instance, rather than the aamino acids assumed in this work, it is possible to include plausible predecessors of these molecules, as well as their peptide products (see Taillades et al., 1998). Similarly, the theory does not address the prebiotic metabolic pathways in which template-directed reactions and ‘‘memory’’ are not involved (see Lazcano and Miller, 1999; W.achtersh.auser, 1992a). 3.3. Specific requirements of the ETIF theory Mechanistic prerequisites for specific scenarios are model-dependent. For instance, the main primordial energy sources of the ETIF theory are thermal and chemical, that of the iron–sulfur world (W.achtersh.auser, 1988) is chemical, whereas that of the primordial vesicles according to Morowitz (1992) is solar. Accordingly, the energy utilization system in each of these theories is different. The specific prerequisites of the ETIF scenario (Table 1) are described in terms of the initial geochemical features of the relevant environment, the central reactions of the organic molecules, and the products of their interactions. Given these prerequisites and the dynamics of the fluctuating environment, as well as plausibility of condensation reactions under these conditions, the emergence of feedback loops with their associated TSD syntheses is assumed to take place (Table 2, Fig. 1). These include information manipulations, such as exemplified in polymerization (see also James and Ellington, 1999), autocatalysis, replication and translation reactions. Additional attributes of life, as well as their evolution can be gradually incorporated into the Table 2 Central molecules and their reactions and functions in the catalytic takeover process of the ETIF theory. The initial mineral catalysts are taken over by non-directed (ND) peptide catalysts, and these are taken over by template-andsequence-directed (TSD) peptide catalysts Catalyst

Mineral catalyst

Non-directed peptide catalysts

TSD peptide catalysts

Molecule

Reactions

Proto-RNA

ND synthesis and elongation (lowest efficiency) ND replication (none)

ND synthesis and elongation (low efficiency) ND replication (sporadic)

TSD synthesis and elongation (high efficiency) TSD replication (high efficiency)

Proto-mRNA

Translation (none)

Translation (sporadic)

TSD translation apparatus (high efficiency)

Proto-tRNA

ND loading of amino acids (none)

ND loading of amino acids (sporadic)

TSD loading of amino acids (high efficiency)

Peptides

ND synthesis (lowest efficiency)

ND synthesis (low efficiency)

TSD synthesis (high efficiency)

Functions

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Fig. 1. A scheme of two primordial sub-cycles of the TSD system with their feedback-loop systems according to the coevolution theory (Lahav and Nir, 1997; Nir and Lahav, 1997). Homochiral TSD peptide catalysts are represented by P, where P1=primordial peptidyl transferase; P2=primordial RNA polymerase; P3a=primordial amino-acyl tRNAsynthetase (specific for amino acid a); P3b=primordial amino-acyl tRNA-synthetase (specific for amino acid b). (A) Translation cycle. (B) Replication cycle. The two cycles are combined to form a feedback loop.

primordial genetic ‘‘memory’’ of the system, which is essentially proto-mRNA. The close . relationships between proto-mRNA and proto-tRNA was addressed, for instance, by Moller and Janssen (1990, 1992) and Lahav (1991). These attributes are based on the chemical reactions, processes and cycles of organic molecules}the predecessors of the primordial biochemical metabolic pathways, which are gradually incorporated into the ‘‘memory’’ of the evolving systems under consideration.

3.4. Centrality of short organic catalysts The suggested involvement of organic catalysts in prebiotic organic reactions according to the ETIF theory will now be addressed, expanding its context to include also the evolutionary role of this involvement and the emergence of bio-homochirality.

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Historically, the possible role of primordial enzymes has been approached predominantly by two opposing strategies, namely, the ‘‘all or none’’ and the ‘‘all and only all’’ schools, both of which represent the extreme sides of the spectrum of prebiotic research strategies. According to the first strategy (the complementary-strand formation experiments of Orgel’s school; see Orgel, 1992), enzyme syntheses are considered prebiotically implausible; therefore prebiotic templatedirected reactions of RNA strands are studied without catalysts, by means of condensation of the activated complementary RNA building blocks. According to the latter strategy (Kauffman’s school: Kauffman, 1993; see also Maynard Smith and Szathmary, 1995; Segre and Lancet, 1998) a whole array of interacting molecules is needed in order to establish a catalytic set and reach the ‘‘phase transition’’ in which life ‘‘crystallizes’’. Rather than lack of prebiotic organic catalysts according to Orgel’s school, or abundance of such catalysts, according to Kauffman’s school, the coevolution and its successor the ETIF theories are strategies of ‘‘just enough’’ prebiotic catalysts for the emergence and establishment of the first feedback loops and their TSD systems. As suggested earlier (see Ninio, 1979; White, 1980; de Duve, 1991; Lahav, 1991; Lahav and Nir, 1997) these catalysts were most likely short peptides. Furthermore, in view of the selectivity characterizing enzymes and ribozymes, it is reasonable to assume that the presumed prebiotic short catalysts are also characterized by crude selectivity. In the following section we discuss the possible role of short catalytic peptides in the transition from inanimate to animate matter. The possible role of ribozymes in the latter transition can be derived similarly and will not be addressed in detail. The ETIF model is general enough to be operated flexibly with regard to its molecular constituents, provided the basic attributes and reactions of these constituents are preserved. For instance, metal ions are assumed to be involved in catalytic processes. However, it is plausible to incorporate into the coevolution and the ETIF scenarios small organic molecules such as amino acids (Bar-Nun et al., 1994; Roth and Breaker, 1998; Rode et al., 1999; Suwannachot and Rode, 1999) acting as catalysts or cofactors in some catalytic reactions. By the same token it is possible to increase the catalytic repertoire of the chemical systems under study by introducing protoribozymes and cofactors based on nucleotides (see Giver et al., 1994; Joyce, 1998). Supported by recent experimental work regarding the role of cofactors in RNA catalysis, the synthesis of peptide cofactors by RNA, and their possible role in translation, Joyce (1998) alluded to the central role of peptide catalysts in the origin of life; his note regarding the possible catalytic involvement of peptides in prebiotic evolution is, in fact, included in the central role of short peptides suggested earlier in a more detailed way in the framework of the autogen (White, 1980) theory and the coevolution theory (Lahav and Nir, 1997), as well as in the present ETIF theory. It is noted that preliminary computer model calculations of the coevolution theory (Nir and Lahav, 1997) suggest a considerable robustness with regard to changes in environmental parameters, such as reactant concentration and temperature. Thus, in spite of lack of details regarding the mechanisms by which organic catalysts and cofactors may affect the environmental landscape in which the feedback system can function, a wide range of their variations can sustain the evolution of the chemical entities under study. Early studies of catalytic properties of both linear and cyclic polypeptides and the role of imidazole and histidine in various model reactions were reviewed by Royer (1980). In these early works it has been established that the catalytic activity of linear peptides cannot be accounted for by a simple combination of the relevant properties of their amino acids.

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There are very few experimental observations regarding short catalytic peptides, i.e., 3–10 amino acid residues, in the origin-of-life literature. It is accepted by many investigators that the catalytic activity of small peptides is rather low. Moreover, in many cases such an activity could not be detected. The lowest limit of the size of these catalysts is one amino acid. Thus, Bar-Nun et al. (1994) observed catalytic activity of free amino acids in various reactions, where iron ions were implicated in the catalytic reaction. White and Erickson (1980) described the catalytic activity of histidyl-histidine in the presence of kaolinite clay mineral in peptide bond formation and under fluctuating environment conditions. More recently it was observed by Suwannachot and Rode (1999) that some amino acids (as well as other compounds), most notably glycine, diglycine and histidine, can catalyze the formation of various homopeptides in a fluctuating environment and in the presence of high salt concentrations. These observations support an earlier postulate regarding the important role of soluble-salts-rich fluctuating environments (Lahav and Chang, 1982). It should be noted that the great majority of catalytic activity experiments were carried out with little or no relevance to a plausible model of the biogeochemical conditions on the primordial Earth. Experimental work under prebiotically plausible conditions includes the involvement of metals such as Mg, Zn, Mn and Fe, as well as the presence of adsorbing surfaces and hydration– dehydration processes. A recent study on the effect of water concentration on foldase activity in polypeptide conformational rearrangement (Xu and Cross, 1999) seems to suggest that the fluctuating environment can provide rather versatile conditions for a variety of prebiotic catalytic reactions. Therefore, lack of catalytic activity of small peptides in solution does not necessarily mean that such peptides cannot function as primitive catalysts under plausible primordial Earth conditions. It is thus noted that the possible role of small organic catalysts, and especially short peptides, has hardly been investigated. Consequently, it is hard to specify what we mean by ‘‘short catalytic peptides’’. Moreover, according to the ETIF theory peptides constitute a library of molecules of varying compositions and lengths. And since peptide bonds can be formed between separate amino acids as well as between amino acids and peptides, or between peptides and peptides, it is suggested that the size distribution of prebiotic peptides is likely to have been considerable. Indeed, following the establishment of TSD syntheses, ligation of peptides would be an important mechanism for the evolution of novel catalysts. As discussed below, in spite of these difficulties it is possible to gain some insight into the problem of the size of the first catalytic peptides, which are central to our theory. For simplicity, we have assumed in our computer model (Nir and Lahav, 1997) two amino acids (presumably glycine and alanine; see below) and catalytic tri-peptides. Presumably, the likelihood of finding catalytic peptides in a library of these molecules increases with peptide size. Recent studies suggest that short peptides of about 6–8 amino acid residues not only can be produced in the laboratory under ‘‘prebiotic’’ conditions, but also may be significant from the point of view of peptides evolution. Thus, using a reaction cell that simulates thermal condition near hydrothermal vents (Imai et al., 1999a, b; Ogata et al., 2000), Matsuno and his group (pers. comm.) have recently observed octaglycine in this reaction cell. In earlier attempts to synthesize peptides under a fluctuating environment and in the presence of kaolinite clay mineral (Lahav et al., 1978) peptides up to hexaglycine were observed. In all these condensation experiments the yield was in the range of 1% or less of the total concentration of the amino acid under study. Presumably, only a part of these peptides can catalyze the reactions under study.

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3.4.1. Merging of top-down and bottom-up strategies: small peptides Until recently, the main interface between the top-down and bottom-up research strategies was the hypothetical meeting zone between the LUCA and the beginning of the phylogenetic tree. Ideally, this zone represents evolutionary continuity. Methodologically, this interface is implied, based on top-down (see, for instance Di Giulio, 1999; Schwabe, 1985; Trifonov and Bettecken, 1997; Woese, 1998) and bottom-up considerations (see for instance, de Duve, 1991; Lahav and Nir, 1997; W.achtersh.auser, 1988, 1992a, b). A complete merger of the two research strategies would be obtained when the full history of all the chemical entities involved in the transition from inanimate matter to the LUCA, and their dynamics, would be reconstructed. The questions we ask now are not limited to the emergence of the LUCA anymore. Rather, our horizon has been expanded to include also the transition from inanimate to specific evolutionary stages of animate matter. And even though complete detailed reconstruction may never be achieved, several elements of this historical continuum can now be suggested. The above amino acid condensations are typical bottom-up experiments, where the amino acids under study are selected by the experimentalists by using both top-down and bottom-up considerations. Various evolutionary aspects of proteins have recently been interpreted (Berezovsky and Trifonov, 2001) according to a top-down approach that seems to complement the experimental results of the above bottom-up strategy. According to the latter investigators the primordial stage of peptides in their evolutionary pathway is characterized by short peptides in the range of 6–8 amino acid residues. In view of these results it seems that the assumption of short catalytic peptides according to White’s (1980) autogen theory and Lahav and Nir (1997) and the Nir and Lahav (1997) coevolution theory is quite plausible. Furthermore, the evolutionary stage of short catalytic peptides consists the fusion zone between the top-down and bottom-up research strategies. Hence, the gap in our knowledge with regard to the synthesis of prebiotically plausible catalytic peptides calls for the establishment of a new generation of laboratory experiments. The size distribution of the populations of catalytic peptides of our model system, like the scenario itself, can be explored experimentally; until this is done, we shall resort to our computer model, which illustrates numerically trends and stages in early development of feedback loops and TSD systems. It should be noted that in addition to size, the solubility of the peptides under consideration is also of great importance in the study of their prebiotic role, as further discussed below.

3.4.2. Supporting evidence from other research areas It is of interest to examine other scientific disciplines in the framework of which catalytic peptides have been studied. This brings one to recent developments in the attempts to mimic and design proteins, and especially, to the design and synthesis of small catalytic peptides and antibodies. Indeed, in spite of the difficulties in the de novo-designed peptides as biocatalysts (see Corey and Corey, 1996), considerable research work has been dedicated to this problem. The de novo design of proteins and peptides has enhanced the challenges of controlling various properties of these molecules (Tuchscherer and Mutter, 1995), including protein folding (Bryson et al., 1995), chemical nature of binding sites (Regan, 1995), the production of synthetic peptide catalysts (Hahn et al., 1990), synthesis of peptide complexes with catalytic properties (Quemeneur et al., 1998), minimization of a polypeptide hormone (Li et al., 1995), catalytic antibodies (Tawfik et al.,

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1994), development of enzymic inhibitors (Katz et al., 1998), and directed evolution of new catalytic activity (Altamirano et al., 2000). These developments are encouraging, since they are likely to motivate researchers to explore the catalytic potential of short organic molecules in prebiotically plausible reactions. 3.4.3. Specificity and selectivity A central aspect of specificity is the discrimination between several substrates competing for the same active site. The most relevant example of specificity of our coevolution and ETIF scenarios is that of aminoacyl-tRNA synthetase and its specific amino acid and particular tRNA, in a mixture of various amino acids and tRNAs (Fersht, 1985). Similarly, a primordial polymerization catalyst for proto-RNA, whether a proto-ribozyme (Giver et al., 1994; Biebricher, 1994), a short peptide, or a combination of these two molecular species, is likely to exert a slight selectivity on its substrate (Wank et al., 1999). The coevolution and ETIF theories suggest the initial involvement of short, non-templated organic catalysts (peptides as well as primordial nucleic acids or their predecessors) in the prebiotic scenario, followed by a takeover process by TSD reactions in the framework of feedback-loop systems. The selective influence of the feedback loops with their TSD systems is thus assumed to be effective in differentiating between various chemical species as well as between stereoisomers, as further discussed below. Furthermore, according to the coevolution model (Nir and Lahav, 1997) it is sufficient to have small catalytic enhancements of the reactions under study in order to initiate and maintain feedback-loop systems with their TSD syntheses. The detailed mechanism of stereospecificity, including considerations of the minimum size of the first organic catalysts, depends on the molecules involved and cannot be fully understood at present, because of the complexity of this system. The fluctuating environment is characterized by strong and constantly varying interactions between the organic molecules under consideration, as well as interactions between them and their environment. Therefore, whether one uses the threepoint attachment model of enzyme–substrate interactions (see Stryer, 1995, p. 521) or the fourlocation model of these relations (Mesecar and Koshland Jr., 2000), these interactions are likely to be affected by the specific parameters characterizing the fluctuating environment. Based on the observed catalytic reactions of minerals in various ‘‘prebiotic’’ organic reactions characterizing fluctuating environment (see, for instance, Bujdak and Rode, 1999; Lahav, 1994, 1999; Suwannachot and Rode, 1999) it is assumed that the feedback loops under consideration are likely to be formed under these conditions. The advent of feedback loops with their TSD reactions thus signifies the beginning of a new evolutionary era, where both the size, catalytic activity and specificity of these catalysts are not only augmented, but also coded in the primitive ‘‘memory’’ of the system, which is proto-RNA. The coevolution and the ETIF theories imply that during the TSD takeover the initial small advantage of the TSD system increases rather fast, culminating in a complete takeover of the entire molecular population under consideration. 3.4.4. Bio-homochirality takeover For simplicity, homochiral building blocks of the two central biopolymers, namely, templates on the one hand, and catalysts on the other, were assumed in the development of the coevolution theory (Lahav and Nir, 1997; Nir and Lahav, 1997). As discussed below, this assumption is not

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needed according to the present theory, since the emergence of primordial feedback loops with their TSD systems entails the emergence of bio-homochirality. The origin of biological homochirality has been one of the most controversial topics in the search of the origin of life. Didactically, symmetry breaking could have taken place either prior, or during, or after the emergence of life, where it is followed by chiral amplification and chiral expansion in the biological systems under consideration (see Bonner, 1995; Brack, 1998; Goldanskii and Kuzmin, 1991; Cronin, 1998; Navarro-Gonzalez et al., 1993; Popa, 1997; Schwartz, 1998; W.achtersh.auser, 1992b; Wald, 1957). It is convenient to divide the published theories, scenarios, or experimental systems designed to explore the origin of biohomochiralities, into two groups, external and internal, as follows: 3.4.4.1. External theories. In this group the mechanism involved in the symmetry breaking of a racemic solution of the relevant building blocks is external, namely, it affects the environment of the entities under consideration. Scenarios based on external agents include a variety of mechanisms such as (i) circularly polarized UV light or enantioselective magnetochiral photochemistry (Rikken and Raupach, 2000) destroying preferentially one enantiomeric form (see for instance, Bailey et al., 1998; Bonner et al., 1999; Greenberg, 1995; Popa, 1997), or (ii) adsorption processes on specific mineral sites in which one enantiomeric form is preferred over the other (see for instance, W.achtersh.auser, 1992a, b; Weissbuch et al., 1994). According to these theories, in the absence of external selective agents, no such externally induced symmetry breaking would occur. On the other hand, in the presence of such agents, an excess or enrichment of one enantiomeric form over the other is achieved. 3.4.4.2. Internal theories. The mere enrichment by one enantiomeric form, i.e., due to selective destruction or adsorption, should be supplemented by a chemical mechanism which would carry out and bring to a completion the amplification process. Thus, a system containing an excess of one enantiomeric form over the other would become homochiral only if it invokes an effective mechanism by which this excess would lead to homochirality. The involvement of internal agents implies scenarios based on selection processes performed by the involved molecules themselves (Bolli et al., 1997). According to the ETIF theory chiral selection and amplification start simultaneously with the establishment of the feedback loops with their TSD systems and result in homochirality of peptides and proto-RNA molecules. It should also be noted that shape chirality is a continuous structural property (Zabrodsky and Avnir, 1995; Keinan and Avnir, 1998), whereas biological homochirality is normally assumed to either exist or to be absent in systems containing amino acids and nucleic acids. The present discussion focuses on the emergence of biohomochirality without introducing the continuous structural feature of chirality. 3.4.4.3. The case of glycine. It is assumed that during the stage of non-directed syntheses and later on}until the evolution of metabolic cycles involved in the syntheses of amino acids}the content of glycine in primordial peptides reflected their concentrations in the microenvironments under study (see Trifonov, 1999, 2000). Based on many laboratory experiments, mostly of the Miller–Urey kind, as well as on meteorite analyses, it is generally accepted that the prebiotic environment was relatively rich in glycine and to a lesser extent in alanine (for recent reviews see

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Cronin, 1998; Miller, 1998). The concentrations of additional amino acids were much smaller. Moreover, the amino acids in these systems were racemic, except for glycine, which is achiral. The ETIF theory does not assume one specific chemical mechanism for the synthesis of the building blocks of peptides. Rather, it can be adjusted to a variety of such mechanisms. By assuming a fluctuating environment, and since the non-directed peptides formed are suggested to reflect the concentrations of the amino acids in this solution, these peptides are expected to be rich in glycine and alanine. Glycine is of special importance in the ETIF theory. Because of its high water solubility, its dissolution–precipitation reactions in the fluctuating environment follow the hydration– dehydration cycles, thus undergoing redistribution quite regularly in this system, where it is condensed with available amino acids, or peptides, to form peptide bonds during the dry stage (Lahav et al., 1978; Lahav and Nir, 1997; see Suwannachot and Rode, 1999). The size and composition of these peptides reflect the properties of the fluctuating environment and its main amino acids. Glycine is not only the major amino acid constituent of the fluctuating environment. It is also the most soluble amino acid, implying high concentrations of this amino acid during the dehydrating period. Taking into consideration also the importance of soluble constituents and surface density of adsorbed amino acids in condensation reactions occurring in the fluctuating environment (Lahav et al., 1980), these considerations corroborate with the suggestion that glycine (and to some extent also alanine, see below) would be the most prevalent amino acid in the primordial peptide populations under study. Indeed, the high percentage of glycine in ancient proteins (Trifonov, 2000) seems to accord with these considerations. The preservation of the high glycine content in ancient proteins is a remarkable demonstration of biological conservatism. Moreover, as noted by Trifonov (2000), the glycine content of the primordial proteins under investigation was higher than that of their extant successors. 3.4.4.4. Symmetry breaking during synthesis of small glycine-rich peptides. The involvement of catalytic peptides in the selection of reactants according to the ETIF theory suggests that should they be homochiral from the very beginning, the process of TSD formation would be greatly facilitated. How can homochirality of peptides be formed in a fluctuating racemic environment during the establishment of feedback loops and TSD systems? The above discussed dominance of glycine in both the prebiotic environment and primordial peptides may have far reaching consequences in the condensation process of this and other amino acids. For simplicity, we assume the synthesis of only glycine–alanine peptides; additional amino acids are expected to behave like alanine in this respect. Since each of the alanine enantiomers acts as an individual chemical constituent, its concentration in the fluctuating environment, namely}both in the aqueous solution of the hydrated state, on the mineral surfaces during the dehydrated stage, and in all the intermediate stages between these states}would be half the total alanine concentration. Using a simplified model, in which the incorporation of the three kinds of amino acids into peptides during the dry stage of each wetting–drying cycle is proportional to their concentrations, a unique situation is expected, namely: when the peptide formation reaction starts from the first two amino acids and proceeds with the addition of one amino acid at a time, some of the peptides thus formed would be homochiral, in the form of either glycine–(d-alanine) or glycine–(l-alanine). For instance, consider a system where condensation reactions between amino acids are catalyzed by minerals in accordance with the coevolution theory, without any

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preference for a specific pair of amino acids. In the presence of the same concentrations of glycine and d; l-alanine in the ambient solution the probability of having a homochiral peptide (either d or l) is given by ! ! L L X X L L i ðLiÞ Pd Pg Pil PðLiÞ þ ; ð1Þ g i i i¼1 i¼1 where L is the number of amino acid residues in a peptide, Pg the probability of finding glycine when randomly selecting an amino acid in this peptide (excluding polyglycine), Pl the probability of finding l-alanine when randomly selecting an amino acid in this peptide and Pd the probability of finding d-alanine when randomly selecting an amino acid in this peptide. ! L L! ¼ number of possible combinations of i identical objects in L position: ¼ i!ðL  iÞ! i Thus, in the presence of the same concentrations of glycine and d; l-alanine, the probability of finding homochiral glycine–alanine peptides (either d or l) of three amino acid residues would be 0.65 whereas that of 10 amino acid residues would be 0.11. Presumably, some of these glycine-(dalanine) or glycine-(l-alanine) homochiral peptides would be able to serve as catalysts, either with or without the help of metals such as Mg and Fe, as well as simple cofactors. Obviously, in a more realistic system the proportion of each of the enantiomers in these peptides would be a function of additional parameters. For instance, glycine is the most active amino acid studied so far in ‘‘prebiotic’’ condensation reactions (Bujdak and Rode, 1999). It is assumed that these extra effects would not change the fundamental conclusion of symmetry breaking by some of the glycine-rich peptides. Because of the low activity of glycine in catalytic reactions, pure polyglycine could have been involved in structural roles. However, homochiral glycine–alanine peptides are expected to be more chemically active than polyglycine. Some of these homochiral peptides can serve as catalysts, inspite of the low activity of glycine in biological catalytic reactions. Their ability to select the appropriate chiral building blocks, i.e., amino acids or proto-RNAs, and catalyze their reactions, is proposed to have been crucial to the evolution of the first feedback loops. Thus, the catalyzed elongation and replication of proto-RNA results in homochiral proto-RNAs. Clearly, these reactions can be studied experimentally: the computer modeling employed in the coevolution theory (Nir and Lahav, 1997) with regard to the ability of TSD systems with their feedback loops to amplify minute concentrations of specific molecules and systems with small evolutionary advantages over other systems, is suggested to be applicable also to the emergence of homochiral catalytic peptides. In concluding this section, it is noted that selection of chiral molecules is assumed to be performed by the homochiral peptide catalysts of the evolving system under study. Extrinsic effects characterizing the external group (see above), i.e., selective destruction by an external agent or adsorption on mineral surfaces, are likely to bring about only an enrichment, not domination, of one enantiomer over the other. On the other hand, chiral molecules competing for substrate molecules and the absence of selection of one of the enantiomers by the template itself (such as in the system studied by Joyce et al., 1984), may impede the chiral selectivity process.

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Finally, it should be noted that the assumption of the involvement of short catalytic peptides in the selection processes is implied in the interpretation of peptide structure considerations (see Section 3.4.1) according to Berezovski and Trifonov (2001). Thus, the suggested role of short, homochiral glycine-rich peptides not only provides a mechanism for the selection process, but also accords with the principle of biological continuity, according to which the role of (short) peptides in the emergence of life is as old as life itself. In other words, the ETIF theory suggests that the emergence of homochirality and life are inseparable. 3.5. Emergence of the genetic code We are now in a position to focus on the two specific amino acids, glycine and alanine, which are assumed to have been involved in the transition from inanimate to animate matter. This conclusion immediately implies the first pair of codons, i.e., GGC and GCC for glycine and alanine, respectively (Eigen and Schuster, 1978). The relations (both in laboratory experiments and observation of extraterrestrial sources of these compounds; see Cronin, 1998) between structural and compositional considerations and prebiotic syntheses of amino acids suggest, according to Eigen and Schuster, ‘‘that assignment of codons to amino acids actually followed the abundance scale’’. In other words, the most prebiotically abundant amino acids, i.e., glycine and . alanine, were the first to be ‘‘recruited’’ to life. Similarly, Moller and Janssen (1990, 1992) differentiate between ‘‘primordial amino acids’’ which have been formed in laboratory experiments under plausible prebiotic conditions, on the one hand, and amino acids that are not formed under these conditions, on the other. According to these authors, the first group left its imprint in the form of a prototype code for these primordial amino acids. These ideas have been also adopted by other researchers (see Kuhn and Waser, 1994; Trifonov, 1999, 2000). Moreover, amino acids and proto-RNAs are connected by the genetic code and thus, according to the ETIF theory, the recruitment of glycine provides not only the mechanism of biohomochirality emergence, as discussed above, but also the emergence of the genetic code. Thus, the interactive top-down and bottom-up strategies regarding glycine achirality and abundances, both in the prebiotic environment and ancient proteins (Trifonov, 1999, 2000), merge into a unified pathway suggesting a catalytic role of short, glycine–alanine-rich homochiral peptides in the transition from inanimate to animate matter. This then implies continuity between the bottom-up formation of short, catalytic and homochiral peptides rich in glycine and alanine on the one hand, and extant, homochiral ancient proteins, rich in glycine and alanine (Trifonov, 2000), on the other. Furthermore, the same top-down arguments can be used for the identification of the first codons (Trifonov, 1999; Trifonov and Bettecken, 1997) and the primordial catalytic peptides involved in the evolution of the first living entities. The above merging of the top-down and bottom-up research strategies is just another point of unification of the two oppositely directed approaches. Additional points of merging need models describing the relations between specific primordial functions and known features of involved molecules and environments. For instance, according to the coevolution and the ETIF theories the three primordial functions of the first feedback loops are (i) peptide bond formation, (ii) loading of amino acids on proto-tRNA, and (iii) proto-RNA replication. It is suggested that the first catalysts involved in these reactions were homochiral glycine–alanine-rich peptides, with possible additional amino acids in considerably lower concentrations. If this is true, then

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conserved sequences of the catalytic sites of the extant enzymes performing these functions, as well as their 3D structure, are suggested to be explored, and may be identified. It should be noted that the present model with its three fundamental reactions is a schematic description of the central reactions of the coevolution and ETIF theories. It is expected that these reactions would be sub divided into additional reactions, as their study progresses. 3.5.1. Merging of top-down and bottom-up strategies: the emergence of the primordial genetic code The suggestion of feedback loops involvement in the primordial TSD synthesis of novel catalytic peptides implies the emergence of an ad hoc genetic code. Since this feedback loop must reflect properties of the environment under consideration, the ETIF theory leans on both the bottom-up and top-down research strategies of scenario reconstruction. In other words, the reconstruction of the establishment of a code, which combines the proto-mRNA, proto-tRNA and the amino acids under consideration into one functional/operational system, integrates these two oppositely directed scenario-building approaches into a continuous scenario (for recent uses and reviews of these attempts see Davis, 1999; Di Giulio, 1999; Kuhn and Waser, 1994; Trifonov, 2000). The ETIF theory addresses various aspects of the origin of the genetic code, i.e., the involvement of proto-RNA molecules and proto-aminoacyl-tRNA synthetases, their sequences and evolutionary history, their chemical and physico-chemical properties and evolution, and the characteristics of the involved environment. The present discussion focuses on general considerations of the establishment of the codons of the first amino acids of the genetic code in the framework of their environment. It is noted that theoretical models which do not involve geochemical aspects of the relevant environments (see also, for instance, Knight et al., 1999; Di Giulio, 1997, 1998, 1999, 2000; Freeland et al., 2000; Lehmann, 2000), imply either that their origin of the code came after the origin of life or that they have yet to be adjusted to their relevant geochemical and geophysical conditions. This is of special importance with regard to optimization studies on the evolution of the genetic code (see, for instance, the debate between Di Giulio, 2000 and Freeland et al., 2000), since involvement of the environment may introduce new parameters to these calculations. Additional central aspects of the role of the environment in the emergence of the genetic code, such as the effect of the thermal history of primordial life on the incorporated amino acids (Trifonov, 1999, 2000), will not be elaborated here. The theories suggested so far for the origin of the genetic code may be divided, somewhat schematically and arbitrarily, into two groups, according to the biogeochemical model involved in the synthesis of the building blocks of biochemistry or their predecessors, namely, (i) primordial metabolism and (ii) prebiotic soup theories. It is noted that theories dealing with physico-chemical considerations}mainly hydrophilicity–hydrophobicity of amino acids, and their possible connection with the genetic system, are found in these two groups (see below and Lehmann, 2000). 3.5.1.1. Primordial metabolism theories. These theories are based on the most ancient metabolic cycles in extant organisms, which supply the central building blocks of biochemistry. By applying the biological continuity guideline it is suggested that these metabolic cycles, or their ancient predecessors, also supplied the central building blocks of the primordial living entities under

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consideration, some time after the establishment of the origin of life processes (see Davis, 1999; Knight et al., 1999; Taylor and Coates, 1989; Wong and Tze-Fei, 1975, 1976, 1980). The main stages of the code evolution and their timing according to these theories were estimated by Davis (1999) from comparative path lengths in the central (and most ancient) metabolic cycles, using a number of assumptions the validity of which has yet to be established. His codon tree is rooted in the four N-fixing amino acids aspartate, glutamate, asparagine and glutamine. Davis’ theory is based on extant metabolic cycles and molecular biology, where its central cycle, namely, the reductive citric acid cycle (RCC), is assumed to be related to the W.achtersh.auser’s RCC. However, in the latter cycle, which is assumed to form spontaneously on pyrite surfaces under the conditions of the deep hydrothermal vents, the emergence of translation is a late development and does not harbor the mechanism of template-and-sequence-directed (TSD) system. This theory will not be discussed here. 3.5.1.2. Soup theories. The original use of this title was assigned to the prebiotic sea, according to Oparin and Haldane. In the present discussion this group of theories focuses on other environmental niches, i.e., fluctuating environments or porous minerals, with the relevant mechanisms for the synthesis of the building blocks. In other words, these theories are based on prebiotic terrestrial syntheses according to Urey and Miller on the one hand (see Eigen and Schuster, 1978; Eigen and Winkler-Oswatitsch, 1981a, b; Kuhn and Waser, 1994; Miller, 1998; Trifonov, 1999), and import of extraterrestrial organic compounds, on the other (see Cronin, 1998). A central feature of the theories of this group is the abundance of glycine and alanine (see above), whereas the concentrations of additional amino acids are assumed to be considerably lower. As suggested earlier, glycine and alanine are assumed to have been the first coded amino acids. Unlike Davis’ theories which is based on primordial metabolism scenarios, the ETIF theory deals with the transition from inanimate to animate matter and beyond, including the emergence of homochiral TSD peptides and proto-RNAs, with their feedback loops and primordial genetic code of the major amino acid products of prebiotic synthesis, namely, glycine and alanine. More generally, the ETIF scenario provides a mechanism for the synthesis of the primordial TSD catalysts, templates and metabolic cycles and thus}the utensil for the evolutionary process in general. The ETIF theory postulates that the first catalytic peptides in the evolutionary pathway leading to the primordial living entities were glycine and alanine-rich molecules. This then implies not only bio-homochirality (see above), but also glycine and alanine as the main constituents of the first catalytic peptides. Implied in this interpretation is the assumption that later evolutionary stages were characterized by a takeover process, where the biosynthesis of amino acids, as related to the RCC (and later}also to the citric acid cycle), became the dominant mechanism of amino acids supply. 3.5.1.3. Temporal order of amino acid recruitment. The apparent discrepancy between the above two theories with regard to the order of codon capture by the first amino acids has yet to be resolved. Indeed, it is suggested that the emergence of the first coded amino acids according to the ETIF theory on the one hand, and according to Davis’ (1999) theory on the other, are not necessarily mutually exclusive.

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According to the ETIF theory the supply of amino acids in the first stage of the emergence of living entities is by geochemical processes in the environment. Accordingly, the main constituents of the catalytic peptides of the primordial living entities were glycine and alanine (see above). Gradually these entities evolved metabolic cycles, where a takeover process of ‘‘geochemical amino acids’’ by ‘‘metabolic amino acids’’ was established. Thus, self-supply of the first coded amino acids glycine and alanine was obtained soon after the emergence of the first feedback loops and their TSD molecules. On the other hand, Davis’ theory focuses on the order of recruitment of amino acids, based on extant metabolic cycles, which have been preserved in living entities of primitive organisms of the phylogenetic tree. It does not address, however, the mechanism of the emergence of the primordial living entities. Obviously, these metabolic cycles have already been equipped with TSD proteins when the recruitment of Davis’ amino acids took place. Hence they represent a late evolutionary stage that followed the takeover process, not preceded it. To summarize, the apparent discrepancy between the ETIF and Davis’ theories with regard to the order of amino acid coding can be approached as follows: the centrality of glycine and alanine in the first codons according to the ETIF theory reflects the time period and stage of evolution during which amino acids were supplied by the environment. The four amino acids which are the root of the codon tree according to Davis are related to the time period after the takeover of amino acid synthesis by TSD metabolic cycles. The two theories are thus complementary to each other and the order of the first six coded amino acids is glycine, alanine, aspartate, glutamate, asparagines and glutamine. Apparently, this schematic approach should be further refined by . taking into account additional observations, i.e., a larger list of ‘‘primordial amino acids’’ (Moller and Janssen, 1990) and more properties of amino acids (Trifonov, 1999, 2000). A more detailed analysis of the order of amino acid recruitment will not be further addressed now. 3.6. Populations of compartmentalized feedback loops and their TSD systems Compartmentation is essential for the feedback loops and TSD systems under study, the two candidate mechanisms for this process being adsorption on minerals and incorporation within lipid vesicles. Adsorption is an essential feature of the coevolution theory, but due to its shortcomings as a primordial compartmentation mechanism it is assumed to serve as a transitional stage prior to the emergence of lipid vesicles. Moreover, it is assumed that the presence of lipid bilayers in a fluctuating environment would result in encapsulation of TSD systems in lipid vesicles, by a mechanism similar to that suggested earlier (Deamer et al., 1994; Deamer, 1997). The sources of lipid molecules are either prebiotic synthesis (see Deamer, 1997; McCollom et al., 1999), or TSD synthesis by an appropriate metabolic cycle forming a feedback loop at a somewhat later evolutionary stage. Thus, the emergence of feedback loops may be viewed as the emergence of populations of vesicles, some of which being equipped with one or more feedback systems. Such populations are characterized by rather facile mixing of TSD systems or parts thereof inside vesicles, incorporation of hydrophilic peptides inside vesicles, and residence of peptides either inside or on the membrane surfaces. The fusion of two or more such vesicles may result in horizontal ‘‘gene’’ transfer. The latter processes facilitate the establishment of similarity between the compartmentalized TSD systems: small environmental niches inhabited by vesicles encapsulating feedback-loop systems are characterized by substantial similarity. This

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similarity is assumed to be an important attribute, which characterizes the emerging vesicle population also during later evolutionary stages. It seems to corroborate with the polyphyletic attribute of the population of cells characterizing both the last common ancestor (Kyrpides and Ouzounis, 1999; Schwabe, 1985; Woese, 1998), and specific molecules such as tRNAs (Di Giulio, 1999), thus suggesting one more aspect of the continuity feature which is transferred in the primeval reticulated phylogenetic tree. 3.6.1. Influx and efflux through membranes An important aspect of the evolution of the populations of the vesicles under study is the influx and efflux across their membranes. The permeability of these vesicles would depend on the properties of both the diffusing molecules and the membranes. It is expected that as feedback loops with their TSD systems inside a vesicle evolve and increase their size, they are less likely to diffuse outside. At the same time, the selectivity of the vesicle membranes would depend also on the composition of these membranes. Obviously, incorporation of peptides (and later on}of proteins) endows the membranes with selectivity. The membrane selectivity for both influx and efflux diffusion of the primordial vesicles thus evolved with time: the effects of TSD peptides increased with the evolutionary stage of translation and the introduction of new peptides. At the same time, evolutionary changes of the feedback loops and their TSD systems inside the vesicles affected their ability to diffuse out, comparable to the extant differentiation between operational and informational genes according to the complexity hypothesis (Jain et al., 1999). This feature probably started at a much earlier evolutionary stage, presumably in the very first primordial feedback loops. 3.6.2. Horizontal gene transfer and a reticulated phylogenetic tree Horizontal (lateral) gene transfer has been shown to be much more common in early organisms than was believed just a few years ago (see Doolittle, 1997; Jain et al., 1999; Ochman et al., 2000). One possible reason for the excessive gene transfer in life’s early days is that ‘‘. . . molecular machinery for replicating and processing genetic material was still universal. . .’’ (Pennisi, 1999), where organisms ‘‘had not yet evolved the means of repelling alien genetic material’’. Other possible reasons presumably involved flexibility of the primordial cellular membranes. Various aspects of these gene transfer processes, including the role of membranes, have been recently discussed (Heinemann, 1998; Lake and Rivera, 1998; Pennisi, 1999). The environmental aspect of gene transfer processes includes concentration of living entities, i.e., the proximity between living entities as a function of water content of the fluctuating environment, as well as temperature regime. The fluctuating environment thus functions as a large scale, continuous shuffling reactor, where the reactants under consideration can form a large variety of associations and interactions. 3.6.3. Size of vesicles and their molecular populations It is instructive to compare the minimal system capable of initiating and maintaining the feedback loops with their TSD processes under study, with other hypothetical primordial entities such as the protocell (Morowitz, 1992), minimal cell (Fleischaker, 1990; Mingers, 1995), and chemoton (see Szathmary, 1994). The latter constructs are characterized by full-fledged and template-directed primordial systems, with no information regarding their emergence and

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evolution. In contrast, the ETIF model deals with non-template-directed molecular populations giving rise to TSD systems in the framework of feedback loop organization, which in turn are gradually enriched with novel coded attributes of living entities. The latter enrichment process starts with a rather humble beginning, i.e., the presence in close proximity of non-directed prototRNA, proto-mRNA, amino acids (most likely}glycine and alanine) and short catalytic peptides. Thus, the concentrations of the reactants of the first feedback-loop systems, i.e., peptides and proto-RNAs, are very low at first. Furthermore, it is recalled that the unique proto-RNA molecules needed for the initiation of the emergence of TSD system are proto-tRNAs. As shown earlier (Nir and Lahav, 1997) and discussed below, proto-tRNAs may constitute only a small percentage of the total number of the primordial population of proto-RNA molecules. In other words, below a threshold combination of reactant concentration and compartment size, which ensures at least one reactant molecule in one compartment, no feedback-loop system with its TSD constituents would emerge in that compartment. Given the size of the primordial compartment, i.e., an amphiphilic vesicle, there is a threshold concentration for each of the reactants, below which no feedback loop with its TSD system would emerge. Conversely, given the concentrations of reactants, there is a threshold size of compartment, below which no feedback loop with its TSD reactions would emerge. These thresholds are model dependent and are not known at present. They can be determined experimentally, however, at least in principle. 3.7. Mechanism for the incorporation of novel attributes into functioning feedback-loop systems When two or more feedback-loop systems are in close proximity they may interact in various ways, i.e., they can compete with each other over ‘‘nutrients’’, ligate their proto-mRNAs with other strands, exchange similar proto-mRNAs, and share the products of primordial metabolic cycles. These interactions inside compartments and between different compartments are the arena for parallel evolution processes during which novel feedback systems are added to existing ones, thus expanding the repertoire of its primitive genome and the translation system. It is noted that the evolution of the first feedback loops with their TSD systems include acquisition of chemical reactions and cycles, incorporating them into their ‘‘memory’’. Such an acquisition is not necessarily limited to one specific environmental niche with its specific chemical reactions. It may also include an occasional overlapping of two or more specific environments, such as the fluctuating environment on the one hand and a volcanic environment at the Earth’s surface, with its presumed reductive citric acid cycle (see W.achtersh.auser, 1988, 1992a, b; Zubay, 1996), on the other. In view of the suggested short emergence time of the first living entities (see below), effective duration of such an overlapping of two or more environments may be a matter of months or years. Thus, the impact of the environment on the evolution process may leave its marks even upon very short exposure time to environmental changes. Hence, according to the ETIF theory, the primordial living entities are capable of increasing rather quickly their own repertoire of stable feedback loops. In the primordial state of a TSD system, mutations are in fact inevitable, and extra copies of catalytic peptides may also be template-directed synthesized; but as long as such a synthesis is not involved in the formation of a novel feedback loop or increasing the efficiency of an existing feedback loop, no evolution would happen. However, in case one or more of the additional peptides would be able to help catalyzing the formation of a novel feedback loop, or serve as a more efficient catalyst for existing

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feedback loops, either a new or improved function would emerge. When this function enhances the emergence of one or more TSD systems, an adequate new feedback loop may serve as the beginning of a ‘‘factory’’ of genetically encoded chemical reactants. Examples of such an involvement are the syntheses of either new oligomers (such as peptides or proto-RNAs), or molecules involved in new reactions, reaction cycles and functions (including, for instance, energy transfer and redox reactions, needed for the feedback loops, as well as compartmentation). The mechanism of the incorporation of novel attributes is described schematically in the example shown in Fig. 2. The added feature is the production of inorganic pyrophosphate (PPi) by thioesters and inorganic phosphate (Pi) (de Duve, 1998), as described in Fig. 2C. According to the ETIF theory, pyrophosphate serves as proto-ATP in the replication and elongation of proto-RNA molecules. When pyrophosphate production is the limiting step in the operation of the feedback loop and its TSD system, the supply of pyrophosphate by the thioester reaction would benefit the survivability of the feedback-loop system under consideration. Templatedirected peptides P4 and P5, which are the translation products of proto-mRNAP4 and

Fig. 2. A scheme of the addition of one more feedback loop to an existing TSD system (see Fig. 1). The added feedback loop is based on pyrophosphate production shown in the figure (reactions 1 and 2, according to de Duve, 1991).

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proto-mRNAP5 molecules, respectively, happen to catalyze the pyrophosphate production (Eqs. (1) and (2) in Fig. 2). This would enhance the replication and elongation of proto-RNAs in the population of TSD systems of the same compartment, providing some advantage to feedback-loop systems capable of synthesizing these two peptides, thus establishing a novel feedback loop. As shown earlier in our computer model (Nir and Lahav, 1997), a small advantage for one or more feedback loops with their TSD reactions in a population of TSD systems is likely to increase significantly their proportion in the entire population. 3.7.1. Late initiations of novel feedback loops The initiation of the emergence of feedback loops with their TSD systems in a fluctuating environment could have started again and again during the geological time-window in which life is assumed to have emerged. However, the ability of such new entities to compete with older, more evolutionary-advanced systems is assumed to decrease with time, because the new TSD entities ‘‘would be instantly devoured’’ by the old ones, as discussed by Darwin in his famous letter to Hooker (see for instance, Lahav, 1999, p. 33). 3.8. Magnitudes of discrete evolutionary steps Similar to discrete genetic changes in biology, the genetical mechanisms of the primeval reticulated phylogenetic tree encompass steps of different sizes. The smallest discrete genetical unit is one building block of a proto-mRNA. On the other extreme of this scale are primordial horizontal transfers similar to known biological horizontal gene transfer (for recent reviews see Heinemann, 1998; Lake and Rivera, 1998, 1999). Thus, it is not necessary to invent de novo the mechanism of the emergence of each novel function or chemical entity: a central feature of the feedback-loop system, with its built-in autocatalysis, peptide synthesis and variability, is the ability to form additional feedback loops, in the framework of either existing or novel feedback loops. Each of these feedback loops is characterized by a specific novel reaction, thus increasing the chemical repertoire of the whole system. The latter includes metabolic cycles and subsystems, such as the primordial reductive citric acid cycle. Thus the feedback loop with its TSD system can serve as a mechanism for the acquisition of novel attributes, which may be phrased as follows: Emergence of novel coded attributes is a re-implementation of the pattern of establishment of the first feedback loop with its TSD system. Moreover, these considerations are applicable to all the primordial mechanisms of mutation, i.e., point mutations, gene duplication and horizontal gene transfer. 3.9. Biological information and its emergence: basic thermodynamic principles For many authors it seems to be intuitively clear that all biological processes involve a great deal of accumulation, exchange and processing of information. Yet the precise role of information in organic phenomena is still unclear. On the one hand, it is obvious that a great amount of information is passed on from one generation to another through genetic heritage. On the other, information theory itself is at a loss to give an objective measure of information’s value, or even to objectively distinguish between a meaningful message and mere noise (Brillouin, 1956). In what

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follows we shall not try to give a final resolution to this old debate. Rather, we shall try to show how the emergence of life was characterized by an emergence of information processing mechanisms that played a very specific, physically observable role in the evolution of primordial life. We shall first briefly review the basic thermodynamic principles underlying the relations between information and energy. Next we will show how evolution takes advantage of these principles.

3.9.1. Information and energy: physical affinities The second law of thermodynamics obliges entropy to increase in any closed system. This formulation still allows entropy to decrease (or order to increase) in open systems. However, the latter case requires energy dispersal, which would increase the entropy outside the open system, such that the next closed system}i.e., the larger system of which the open system is a part}increases its overall entropy. An apparent contradiction of this principle has been pointed out during the 19th century, giving rise to a paradox known as ‘‘Maxwell’s demon’’. The eventual resolution of this paradox provides an important insight into the nature of evolution, biotic as well as prebiotic, and we will try to apply this insight to our work. Maxwell considered the case of a closed box full of gas in equilibrium, with a partition dividing the box into two, and a small door in the partition. A microscopic demon can increase the order in the gas by opening and closing the door such that all fast molecules concentrate in one-half of the box and all the slow molecules in the other. Macroscopically, the result would be that the gas in equilibrium has been separated to hot and cold gas, enabling the performance of new work. Here, order has markedly increased with the aid of only negligible energy, in apparent defiance of the second law. The resolution of the paradox has enriched physics with a notion whose technological and conceptual importance can hardly be overestimated. Here it is in brief. Although the demon produces order and complexity with negligible input of energy, it must use information about the molecules to be sorted. Since the acquisition, storage and processing of that information must have already taken their thermodynamic costs, there is no violation of the second law. Based on this resolution, Elitzur (1994) has proposed a succinct formulation of the process by which energy is saved in all biological processes: With the use of information, work can be carried out with little energy, but at the right place and the right time. The simplest example is the task of opening a locked door. Breaking the door requires a lot of energy, while opening it with a key achieves the same goal with much less energy. Here, the key contains the necessary information about the door’s complex structure, thereby allowing the careful use of the little energy available to the user at the right spot within the lock’s complex machinery. Information and energy are therefore related in two reciprocal ways: (i) any generation, storage, transmission and processing of information takes its price in energy. (ii) Conversely, with the aid of information, energy can be considerably saved by increasing the precision of the work done by it. In order for these formulations to be useful for a working model, they need to be made quantitative. We shall next briefly introduce the conventional measures of information.

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3.9.2. Information and entropy: mathematical affinities In order to quantify information, information theory takes the thermodynamic concept of entropy as a basis. Entropy’s measure is given by Boltzmann’s equation S ¼ k ln W, where S is the entropy, k the Boltzmann’s constant and W the number of possible microstates. The last term turned out to be equally useful for measuring the inverse of information, namely, uncertainty. Ignorance about a certain state means that many possibilities have the same probability for that state. Information, then, reduces these possibilities, ideally to probability 1 for one of them. This has led Brillouin (1956) to define information, I, as the difference between the initial and final uncertainties. Let there initially be p0 possible states, all equally likely, hence there is no information at this stage, I0 ¼ 0. Having received the information, the number of possibilities drops to p1 . The information thus gained is I ¼ k lnðp0 =p1 Þ ¼ kðln p0 ln p1 Þ:

ð2Þ

Unfortunately, this measure of information, while very useful for communication sciences, is much less applicable for the biologist. For ‘‘uncertainty’’ is a subjective notion, and is of little use when dealing with simple systems that do not use communication at all. Of course, one can use it to measure the amount of information transferred by the DNA from one organism to its offspring. But this technical measure, as pointed out above by Brillouin (1956), cannot distinguish between useful information and mere noise. Given that there are four nucleotides, the information content for n nucleotides DNA segment would be n4 , implying a high information value. But this would equally apply for a random sequence of n nucleotides. We would like to go one step further and argue that, in biology, a more fruitful measure can be given to information’s value. 3.9.3. Information and energy: biological affinities We first note that Maxwell’s demon and the resolution of the associated paradox have a straightforward bearing on all biological processes. All living systems perform work in numerous forms. A closer inspection of these processes always shows the energetic cost of this work to be surprisingly small, even minimal. Let us take a dramatic example. The tiger exerts enormous mechanical force to kill its prey. The cobra, in contrast, kills a prey of the same size by merely spitting into its eye. What is striking in the latter case is the disproportion between the negligible force exerted on the prey and the fatal results. The secret lies in the snake’s ‘‘choice’’ of the appropriate neurotoxin, that matches the prey’s synapses by its uncanny resemblance to its neurotransmitters, and the ‘‘choice’’ of the precise vulnerable point to penetrate the prey’s vascular system (recall that it is the prey’s own vascular system that carries the venom from the eye to the entire nervous system!). In other words, the cobra makes virtuous use of information about its prey’s anatomy and neurochemistry that allows it to save the required energy that the tiger has to ‘‘invest’’ for the same purpose. Here too, the force exerted by venom is literally infinitesimal}of molecular scale}but it is exerted at the right place and at the right time. This suggests an objective, physical measure of information value. When work is performed with the aid of information, the latter value is given by the amount of energy saved by it I ¼ e1 =e2 ;

ð3Þ

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where e1 is the energy that would be needed had the work been performed without the use of that information, and e2 the energy actually used. Thus, the information value of a biological process can be measured by comparing the energy that it takes, with the energy that it would require without the aid of information. In trying to use this approach for actual cases, the information content of various systems has to be compared. The following systems are of special interest, namely, (i) differences between two living organisms, (ii) differences between a living system and a man-made system synthesizing the same products, and (iii) differences between a living system performing a certain chemical reaction and an inorganic system performing the same reaction. An example of the first system is the effect of a single point mutation on a living entity. An example of the second system is the photosynthetic process, which takes enormous energy when performed by present-day technology but negligible energy when performed by a simple plant. The third example, the subject matter of the present work, is the emergence of the first feedback-loop system, with its TSD reactions, according to the ETIF theory. In all these examples, the difference in energy required by the two systems under consideration is due to information stored during the living system’s evolution and development. In the following discussion we focus on the latter system, namely, the emergence of information during the transition from inanimate to animate matter. 3.9.4. Information and the emergence of the first feedback loop For the first feedback loop of the transition from inanimate to animate matter, according to the ETIF theory, a minimum efficiency was required in order to perform the specific reactions needed for its replication and translation reactions. This feat had to be achieved within the brief period during which the feedback loop unit existed before disintegrating. Here too, the formula ‘‘little energy at the right place and at the right time’’ applies. Notice that our first kinds of ‘‘working’’ molecules, according to the ETIF theory, were catalysts and templates: both kinds lower the energy barrier of the reactions under study. These two kinds of molecules are the predecessors of enzymes and nucleic acid templates of extant living entities. The first information involved in the emergence of life, which marked the beginning of evolution process, was accidental information. This term might sound like an oxymoron, yet everyday life abounds with relevant examples. A random sequence of digits, for example, might by pure chance be identical to a secret code. What makes accidental information so unique in the evolutionary process is this. Once the accidental information occurred in a system where it could make difference in the system’s survivability, natural selection processes further improved its performance at this capacity. The mechanism of the selection process, which results in the ‘‘survival of the fittest’’, is the feedback loop. The minimal evolutionary system capable of carrying out the selection process is the feedback loop with its TSD reactions, which serves as the organizational principle of living entities. Biogenesis, according to this formulation, was the first appearance of a feedback-loop system with its TSD reactions, in which genetic information emerged from accidental information. Evolution also involves adaptation, but no adaptation can occur without detailed information about the environment to which the living entity has to adapt. The theory of evolution can thus be reformulated in thermodynamic and organizational terms as follows: evolution is the process by which living entities acquire, accumulate, process and respond to information about the constituents of their own feedback loops and TSD systems, as well as their environment, thus

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increasing their survivability by affecting their replication, translation, chemical cycles and selection reactions. 3.9.5. Structural information content of the first feedback loops We are now in a position to focus on the mechanism of the emergence of the first biological information. Remembering that the emergence of life is characterized by feedback loops, by which information is manipulated, the problem one has to explore is the information content of the first feedback loops. According to the ETIF theory this information is structural, namely, the sequences of the molecules involved in information manipulation. These are the molecules produced by the TSD reaction, i.e., two kinds of proto-RNA molecules (proto-tRNA and protomRNA templates) and catalytic peptides involved in the translation and replication reactions. It is instructive to explore the minimal size of templates and catalysts plus their corresponding information content, of the first living entities according to the ETIF theory. Obviously, the central organic templates and catalysts involved in the emergence of life are oligomers rather than monomers. Moreover, only a small percentage of the total population of proto-RNAs can form the secondary structure characterizing proto-tRNA. As shown earlier (Nir and Lahav, 1997), given catalytic peptides that catalyze the central reactions of the ETIF theory of the emergence of life, proto-tRNA is more likely to become the limiting factor in the closure of the first feedback loops, than proto-mRNA. In order to serve as a proto-tRNA, a proto-RNA molecule must be of a minimal size, in addition to be characterized by a sequence of proto-nucleotides by which its secondary structure is achieved. In other words, both the size and structural information content of proto-tRNA cannot be less than certain minimal values. Thus, with regard to magnitude, the size of a molecule serving as proto-tRNA cannot be smaller than the minimal size of a proto-RNA that can adopt the secondary structure of proto-tRNA. This size may be considered a threshold below which closure of feedback-loop systems would not be possible. Similarly, the information content of a proto-RNA molecule consisting of only one kind of building blocks is too low. If such a strand serves as a proto-mRNA, then the products of a translation reaction, namely TSD peptides, would hardly be able to catalyze more than one reaction. Moreover, in view of the predominance of glycine in the prebiotic environment (see above) such a peptide would probably be made of glycine, and its catalytic activity is expected to be rather low. Thus, at least two kinds of building blocks of proto-RNA are needed. Theoretically, catalytic peptides can also become limiting factors in the closure of feedback loops, if one takes into consideration their size and sequence of amino acid residues. Unfortunately, estimates of threshold values of information characterizing catalytic peptides in the context of their size and sequence, and for given reactions of feedback loops, are premature because of lack of know-how regarding small peptide catalysts (see above). Nevertheless, in view of the facility of the synthesis of amino acids and peptides under ‘‘prebiotic’’ conditions, it is suggested to adopt the working hypothesis that catalytic peptides are less likely to become limiting factors in the fluctuating environment under study than proto-RNAs. Thus, the emergence of the first living entities is equivalent to the emergence of the first feedback loops plus their TSD system, which in turn, is equivalent to the emergence of the first biological information. Of these three qualifications, only information can be quantified. Primordial life can thus be characterized as a feedback loop–TSD system with information content above a certain threshold value. And even though the latter attribute is model-dependent,

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the calculation of its information content can be carried out without the difficulties involved in complex entities such as more advanced stages of living organisms, at least in principle. 3.9.6. Biological information is everywhere Obviously, the Gedanken Experiment described in the discussion of Maxwell’s Demon only demonstrates the importance of information, using a very simple example. Biological information is very versatile and always much more complicated than that demonstrated by the solution of Maxwell’s paradox. The prebiotic processes under discussion provide examples of the diversity of biological information, as discussed now: following the solution of Maxwell’s paradox, it is interesting to consider now the above discussed primordial lipid vesicle, which abides between an extant living cell on the one hand, and Maxwell’s boxes on the other. In such a system the sources of information are more complex and versatile, i.e., because of (i) vesicle’s walls (pore size and physicochemical properties), (ii) involvement of organic molecules in the reactions inside the vesicle, such as in feedback-loop systems, (iii) binding to the inner wall of the vesicle, and (iv) the microenvironment of the vesicles, such as in a fluctuating system. A vesicle is an example of a means to enhance the concentrations of essential molecules in a small volume element. Assume a variety of small molecules and building blocks, i.e., amino acids and metabolites, that can penetrate through the membranes of a given lipid vesicle. At the same time, larger oligomers, such as the ones formed inside the vesicle by the feedback-loop system under consideration, cannot diffuse out through these membranes. Thus, each of these spontaneously formed vesicles works like a pump for the building blocks and stores the products of the feedback loop with its TSD system under study. An added factor is the binding of molecules to the inner walls of the membranes, which results in an overall enhanced concentration of the bound molecules in the volume occupied by the vesicle, while keeping their concentrations in the internal solution smaller than the external solution. 3.10. Primeval reticulated phylogenetic trees and the origin of life Fig. 3 is a scheme (not drawn to scale) of the ‘‘genetic’’ history of populations of entities characterized by feedback-loop–TSD systems undergoing evolution along the vertical time axis, according to the ETIF theory. The large blue spheres designate the formation of feedback-loop systems compartmentalized by lipid vesicles and characterized by the minimum number of the above-discussed attributes of life, at a certain moment on the time axis. The evolutionary pathways emanating from these entities are designated by the somewhat tortuous lines. Some of these lines reach the evolutionary stage of the LUCA, whereas others die out before reaching this stage. Fusion of the lines (dots in the junctions) designates horizontal gene transfer. It is noted that most of the blue spheres starting at a relatively late time die out, whereas most of those who start early reach the stage of LUCA. The population of living entities in Fig. 3 is assumed to inhabit a specific set of ecological niches during the time period starting from the initial establishment of the first feedback–TSD system and extending to the LUCA. The closure of the first feedback loop in each of the compartmentalized chemical entities (designated as blue spheres) is a rather distinct landmark, serving as the beginning of a sequence of feedback loops closure events, which are assumed to have led eventually from the population of these compartmentalized TSD systems to the

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Fig. 3. A scheme of evolutionary pathways of a population of primordial living entities (large, blue spheres).

population of cells serving as the LUCA. During this evolutionary pathway, it is recalled, the closure of a feedback loop is the mechanism by which novel attributes are gained and the primordial evolutionary process proceeds. This implies a gradual enrichment of the compartmentalized entities under consideration by additional feedback loops and coded attributes. The transition from the first TSD systems to the LUCA is thus a continuing process of evolutionary jumps by novel TSD systems and a gradual increase in the number of coded attributes, such as novel templates and proteins, metabolic cycles and information transfer fidelity. 3.11. On the evolution of self-sustained systems Being dependent solely on their environment and its rhythm, the primordial feedback loops with their TSD systems must have been sensitive to environmental conditions. These encompass parameters such as temperature, radiation and water content and their dynamics on the one hand, and supply of organic and inorganic reactants needed for the TSD reactions on the other. Selfsustained assemblies of organic molecules became possible with the advent of feedback-loop systems. This event coincides with the emergence of TSD synthesis, genetics, molecular evolution and metabolic cycles. Moreover, due to the low stability of these primordial feedback-systems on

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the one hand, and their autocatalytic nature on the other (Nir and Lahav, 1997; White and Raab, 1982) it is expected that their emergence was fast (see below). Later stages of evolution of the translation machine and the genetic code were probably a prolonged process (Di Giulio, pers. comm.). The problems of harsh environmental conditions for living entities have been overcome by adaptation through natural selection. Thus, the fragility of the first feedback-loop systems could be overcome by a fast evolutionary acquisition of various essential features almost simultaneously, i.e., catalytic peptides, membranes and the building blocks of the central oligomers of the ETIF theory. It is in this context that the importance of the populations of feedback-loops–TSD systems is central: rather than independent, non-collaborating feedbackloop systems, it is suggested that several such systems evolved simultaneously different feedback loops and different functions, that is, the whole population of such collaborating entities came up every once in a while with various novel functions. Some of these were beneficial, whereas others were neutral and still others were deleterious. In other words, the transition from inanimate matter to the first feedback-loops–TSD systems or population thereof is likely to have evolved in parallel in many primordial compartments, or in populations of such compartments. The latter case involves horizontal gene transfer process, which is assumed to have been very common at this evolutionary stage. Moreover, since these feedback-loop systems must have had a certain degree of variability, the population of such compartments must have been large enough so as to have a reasonable chance to contain the minimal number of novel vital functions, in order to be able to further evolve. The ability of these chemical entities to synthesize the building blocks of their own oligomers and polymers, independent of the primordial supply of such building blocks by the environment, is the beginning of the self-sustained era. Gradually the TSD systems inside the vesicles expanded their repertoire of catalysts, reactants and functions, thus delineating the primeval reticulated phylogenetic tree. This evolutionary pathway culminated in the emergence of the hypothetical last common ancestor, which is a population of primordial cells with considerable similarity (Di Giulio, 1999; Schwabe, 1985; Woese, 1998). 3.12. Fundamental attributes of primordial living entities It is convenient to list, somewhat schematically, the most fundamental attributes of the above primordial chemical systems, which are involved in the transition from inanimate to animate matter, and beyond, according to the ETIF theory. We relate the term function to living entities, as opposed to performance without a purpose-like task characterizing an inanimate system. These attributes are: (i)

Feedback-loop–TSD systems based on complementarity among templates on the one hand and organic catalysts on the other. Basically, this includes replication and translation processes. (ii) Catalyzed reactions directed by recognition and selection, based on structural information accumulated throughout the evolutionary process of the emergence of life. The first landmarks of this evolutionary processes are bio-homochirality, TSD syntheses takeover and metabolic cycles, followed by ever increasing specificity, efficiency and kinds of catalysts.

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(iii) Environmental involvement, including rhythmicity, adsorption, and compartmentation, in the microenvironment of the chemical entities under consideration. Each of these basic attributes by itself is less than what we consider ‘‘extant life’’. It is their collaboration in the framework of evolution that begets extant life, the embodiment of a variety of complex features, including replication, translation, variability, autocatalysis, metabolism, organization, functionality and teleonomy (see below). It should be noted that selection and variability are at the heart of Darwinian evolution. Selection is related to recognition (and hence also to information) and its execution involves catalysis guided by information. Variability among the above primordial evolvable, ‘‘living’’ entities was inevitable and therefore always present. Thus, the prerequisites of Darwinian Evolution, namely, variability and selection, are attributes of the feedback loops under study. Obviously, the two fundamental attributes of the chemical entities under consideration, namely, feedback loops, TSD-informational reactions and catalysis, are the chemical mechanisms of the Darwinian evolution. The closure of a feedback loop therefore marks the transition from a chemical to a functional reaction. The chemical reactions are in the realm of chemistry, whereas the functional reactions are in the realm of biology: it is the purposeful-like behavior of living entities, namely, teleonomy. The emergence of our feedback-loop–TSD systems is thus the beginning of functionality, i.e., the beginning of teleonomy (see below) and the beginning of life. Hence, the feedback-loops–TSD systems of the ETIF theory and their operation may be considered the dynamical principle of the emergence of life. Moreover, it seems that the entire evolutionary process of living entities may be described according to one principle, namely, the gradual addition and development of feedback loops in existing living entities. The feedbackloop–TSD system of the ETIF theory may thus be considered as the organizational units of life. This is further discussed below. 3.12.1. Teleonomy and functionality Teleonomy is an essential attribute of life (Lifson, 1997; Sattler, 1986; see Lahav, 1999 and references therein). The ETIF theory describes the emergence of this attribute, which is the sum of all the functions of discrete feedback loops (TSD systems, whether individuals or fused into bigger entities) resulting from an emergence process by which each of the attributes of life, in their primordial form, is gradually added to the population of TSD systems. Every evolutionary ‘‘innovation’’ is the result of the closure and addition of feedback loops of specific kinds of a TSD system. These feedback loops, with their selectivity and variability, are thus the smallest organizational units of selection. At the same time, these feedback loops are units of functionality, characterizing the teleonomic nature of the primordial evolutionary process. This is reflected by the teleonomic features of the whole, of which these feedback loops are a part. Therefore, the transition from inanimate to animate can be considered as the emergence of functionality, which is equivalent to the emergence of teleonomy. 3.12.2. Evolutionary order of appearance According to the ETIF theory, the first function co-emerging with the first feedback loops was information processing, namely, replication and translation. Because of their fragility, the very survival of the first feedback loops with their delicate networks of reactions meant a rapid

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evolution of various functions almost simultaneously. These functions are essentially metabolic, including the most ancient metabolic cycles, i.e., the reductive citric acid cycle, biosynthesis of amino acids, energy metabolism, and precursors of phospholipids and proto-nucleotides. The emergence of these attributes in a short period of time is much more likely to happen if carried out in parallel. This consideration corroborates with the ETIF description of the first living entities as populations of similar chemical systems. The next relevant evolutionary steps of such populations would be ligation of proto-RNAs, to form a primordial genome with its evolutionary advantages (see Maynard Smith and Szathmary, 1995).

3.13. Comparison with other approaches Except for David White’s (1980) autogen theory, none of the other theories of the origin of life address the emergence of TSD reactions and feedback loops in a specific environment. For instance, the central part of the iron–sulfur theory (W.achtersh.auser, 1988) deals with reaction cycles prior to the stage of the emergence of feedback loops of templated information. Similarly, Morowitz (1992), who suggested lipid vesicles as a suitable microenvironment for the initiation of metabolism and the beginning of life, does not address the emergence of templated information either. Nor does Edward’s universal–ancestral–metabolic-complex address the mechanistic aspects of the emergence of TSD systems. Moreover, the environments characterizing each of the above four theories are rather different from each other, i.e., the fluctuating environment for the ETIF theory, hydrothermal vents for the iron–sulfur world, the sea surface for Morowitz’s floating vesicle, or the sea surface plus pyrite crystals according to Edwards (1996). Indeed, similar considerations regarding additional scenarios and models show not only that they hardly have central features of life in common with the ETIF theory, but also that they cover only a limited number of the attributes of life. Still, it is of interest to mention two more aspects that have been discussed in the literature, namely, simplification and double origin.

3.13.1. Simplification: replicators The importance of simplification upon extrapolating from extant biology to the origin of life has been recognized by biologists long ago. It is the ‘‘how’’ problem that has been controversial among origin-of-life researchers. One simplified theoretical construct is the ‘‘replicator’’. As suggested by the name, this theoretical entity can replicate. According to Dawkins (1989) genes, i.e., DNA molecules, are replicators. Of course, no known gene can replicate by itself; the replication is carried out by a system which includes catalysts. In order to simplify the cellular system such that it would be applicable for the prebiotic environment, various researchers (see Dawkins, 1989) assumed a replicator molecule as a simple system capable of replicating itself. However, except for specific cases of small oligomers (von Kiedrowski, 1986, 1993), no one has succeeded so far to replicate DNA or RNA molecules in the laboratory without a catalyst (see Orgel, 1992; Lahav, 1999, and references therein). Consequently, it is suggested that the simplification involved in the use of the concept of a molecular replicator goes beyond the minimal unit of life and thus does not herald an origin of life scenario simpler than that according to the ETIF theory.

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3.13.2. Simplification: double origin Another attempt of simplification has been suggested by Dyson (1985), following Oparin, in his double-origin theory. According to this theory ‘‘the origin of life is separated from the origin of replication’’, where ‘‘Oparin is describing the first origin of life and Eigen the second’’ (Dyson, 1985). However, the interactions between the polymers of Dyson’s theory, i.e., peptides and nucleic acids, start at a rather advanced stage of their development, where these molecules are rather complex. This means that the implied informational interactions between the peptides and nucleic acids that characterize extant life, i.e., translation, would start at a stage where the polymers are non-template directed. Thus, rather than establishing collaborating sequences of these two polymers from the beginning of their syntheses, Dyson’s theory proposes the beginning of collaboration between established polymers that had been synthesized without earlier interacting with each other. This is unlikely. On top of all this, it is noted that both the replicator and the double-origin theories do not address central problems of the origin of life such as the emergence of homochirality and the role of the environment, which are fundamental to the ETIF theory. 3.13.3. Replicators and units of selection A replicator is usually defined as a molecule capable of ‘‘creating copies of itself ’’ (Dawkins, 1989). Implied in this usage is a replicator according to Orgel’s school. The system approach used in the ETIF theory is based on back-extrapolation from biology. As such, none of its constituents can work alone; replication is performed by a TSD system, which is considered the minimal network of chemical entities and interactions capable of forming an evolvable feedback loop. The system approach considerations may be also extended to the first primordial unit of selection. The biological unit of selection according to Dawkins involves the gene. Following the present approach, the first unit of selection is described as the first sequence of building blocks of proto-mRNA directing, through the TSD system, the first catalytic peptides and proto-RNAs. Hence, the unit of selection may be defined as the first feedback-loop system, with its TSD reactions. 3.13.4. Comprehensive versus partial theories Most published theories of the origin of terrestrial life are limited to a few reactions, with a small number of aspects of this process. For instance: (i) most of the origin-of-life publications so far have been dedicated to prebiotic synthesis of building blocks of biopolymers. (ii) Orgel’s school of thought (Orgel, 1992) focuses on complementary strand formation of nucleic acids. (iii) Deamer’s (1997) school of thought focuses on lipid vesicles, their formation and ability to encapsulate organic molecules relevant to life’s emergence. Obviously, the results of these studies by themselves do not constitute a paradigm of the origin of life, even though they have been an essential step in the search for a general theory of life’s emergence. Thus, among the thousands of papers in the scientific discipline of the Origin of Life, only a few qualify as comprehensive theories. These include the ‘‘Clay world’’, the ‘‘Pyrite world’’ and the present ETIF theories. It should be noted, however, that in principle the partial theories can be further developed to include more and more aspects of life. Obviously, there is no sharp boundary between comprehensive and partial theories of the origin of life. It is noted, however, that the pre-

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paradigmatic stage of the search for the origin of life is reflected by the fact that these comprehensive theories are very different from each other. 3.14. The fundamental unit of life and life’s definition A common denominator for most modern theories of the origin of life, whether explicit or implicit, is the principle of biological continuity. Moreover, it is generally accepted that the deeper the origin of an attribute of life in the primeval reticulated phylogenetic tree is, the more conserved it is (see Lahav, 1999, Chapter 10, and references therein). This then suggests that quite a few of the attributes of the chemical entities characterizing the very beginning of life are likely to have been essentially preserved during the whole history of evolution of life. This viewpoint, which is fundamental to the ETIF theory, thus has a very interesting consequence with regard to a concise characterization, or even definition, of life. As is well known, the multifaceted nature of extant life makes it very difficult, and according to some researchers even impossible, to define life (for recent reviews see Chyba and McDonald, 1995; Lahav, 1999; Luisi, 1998). Indeed, any attempt to define life or living entities depends on the goal of the definition. With the background of the general characterizations of life such as suggested by de Duve (1991), Emmeche (1994) and Lahav (1999), we shall start now with a broad characterization of the central attributes of living entities. 3.14.1. A broad characterization of life The following characterizations are wide enough to touch upon the central attribute of living systems: Metabolism. A living system exchanges, stores and processes matter and energy from its environment. Autonomy. By which it performs work on the environment and on itself. Teleonomy. Which has three major effects on itself: (i)

Feedback. The system perpetuates its own structure and dynamics by countering external changes. (ii) Steady state and reproduction. The system constantly rejuvenates itself by recurrent resetting of all its mechanisms and by countering internal amortization. (iii) Development. The system refines and increases the efficiency of its structure and dynamics. Learning. The system development, ontogenetic and phylogenetic, is utilized by the use of information. Information enables increasing the efficiency of any given work by the use of less and less energy, but more and more accurately at the right place and/or right time. 3.14.2. Historical-mechanistic definitions according to the ETIF theory Remembering that the goal of the present work is to develop a theory of the origin of life, we shall now focus on definitions based on our ETIF theory. A unique feature of this theory is the use of a mechanistic unit of life, namely, the feedback loop with its TSD catalyzed reactions. This fundamental ‘‘building block of life’’ serves as a common denominator for all living forms, both

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extant and extinct, since the emergence of the first living entities from inanimate matter. Moreover, such a definition encompasses the entire history of life. The following three definitions exemplify the flexibility of the definition with regard to one’s ability to emphasize different aspects of the fundamental definition. (1) A living entity is an ensemble of molecules, which exhibit spatial organization and molecular-informational feedback loops in utilization of materials and energy from the environment for its growth and reproduction. While addressing the feedback loop, this definition does not address the main attributes of the feedback loop itself. The following definition is an attempt to focus on feedback-loop systems while characterizing the role of the molecules involved in the living systems and TSD interactions in the context of their environment. According to the ETIF theory, the feedback loops with their TSD systems are the elementary units, or dynamic cyclic components, of life. Hence, the definition of the elementary unit of life, which is also the minimal unit of life, is both a succinct and a fundamental description of living entities. The suggested definition is: (2) A living entity is an ensemble of molecular-informational feedback-loop systems consisting of a plurality of homochiral organic molecules of various kinds, coupled spatially and functionally by means of template-and-sequence-directed networks of catalyzed reactions and utilizing, interactively, energy, and organic and inorganic molecules from the environment. This definition focuses on the fundamental units of life, namely, homochiral feedback-loop systems with its TSD systems. The third example is an attempt to include in the definition the historical aspect of life since its emergence as follows: A living entity is the end product of a continuous succession through time of sequences of feedback-loop systems based on TSD catalyzed reactions since emergence from inanimate matter to the very moment of observation. In other words, each living entity, at any evolutionary stage, is characterized by its evolutionary history, which is given by a progression through time of its feedback loops. The latter definition is more succinct than definition (2) above, since it focuses on the question ‘‘what is a living entity?’’ without addressing the central means by which these molecular-informational feedback loops and their coded forms are formed and function. The difference between these kinds of definitions is related to the ‘‘How?’’ question. Life is the result of the functioning, interacting with the environment, expanding and multiplying of these feedback loops. It encompasses the built-in selection of feedback loops, which result in the survival of the fittest in a population of such entities. Darwinian evolution may thus be viewed as a process resulting in sequences of preserved and linked variable feedback-loop systems. 3.14.3. The emergence of life and its definition are model dependent The above definitions of life are helpful in comparing different origin of life theories. According to the ETIF theory, the emergence of life is equated with the emergence of molecularinformational feedback loops. Other theories of the origin of life, however, define life differently, and therefore the comparison between origin of life theories is not always straightforward. For instance, according to W.achtersh.auser’s (1988, 1992a, b) iron–sulfur world, the beginning of life is

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identical with the formation of his first metabolic cycle and thus represents a rather low degree of molecular organization. Accordingly, the organic entities of his scenario would be included in our definition of life only upon reaching the stage of the establishment of molecular-informational TSD feedback loops. This argument is applicable also for other suggestions, such as the vesicles suggested by Morowitz (1992). 3.15. Time window and duration of life’s emergence 3.15.1. Synthesis and decomposition The balance between formation and decomposition of the molecular assemblies involved in the TSD syntheses affects the emergence time of the hypothetical entities serving as TSD systems with their feedback loops. When the rate of formation is larger than the rate of decomposition a feedback-loop system with its TSD reactions would be likely to emerge. These considerations may be used for the estimation of the emergence time of such feedback loops, if the rate constants of the central reactions of the system under study are known (White and Raab, 1983), as discussed below. 3.15.2. The WHEN and DURATION problems The major aspects of the time factor are the when and duration of the emergence of life process. It is generally accepted that the most ancient indications for life are about 3.8–3.9 billion years old (Arrhenius et al., 1997). The duration of this process, however, has always been a controversial subject; it has been estimated by a variety of approaches (for recent papers and reviews see Lazcano and Miller, 1994, 1996; Miller et al., 1997; Orgel, 1998) and depends, among other parameters, on the definition of life. The first attempt to estimate the time needed for the establishment of the primordial molecular organization similar to our feedback loop was pioneered by White (1980) and White and Raab (1983) in their ‘‘autogen theory’’. The autogen represents a primordial organizational level based on hypercyclic relationships between catalysts and templates; its emergence time was estimated to have been several tens of years. This theory was criticized however (Joyce, 1983) and has not been used since then. 3.15.3. Estimates of duration of life emergence according to the ETIF theory Following White and Raab (1983), the criterion of the ETIF theory for the estimation of the duration of life’s emergence is the time required for significant increase in the concentrations of the synthesized peptide catalysts of the primordial TSD system. This increase corresponds to the closure of the first feedback loops and the concomitant exponential growth of the concentration of catalytic peptides and templates. The computer simulation of Nir and Lahav (1997) yielded concentrations of 10 nM for short peptides after 2.4  104 ‘‘time units’’ or 2  103 days. These numerical calculations considered catalyzed elongation of proto-RNA templates and their replication. A small fraction of these proto-RNA molecules serve as proto-tRNAs, which are capable of being loaded by amino acids. The loaded proto-tRNAs reach by diffusion the template proto-mRNA, resulting in template-directed peptide synthesis, according to an ad hoc primordial genetic code. If some of the resulting peptide molecules can catalyze one or more of the reactions involved in the initiation and maintenance of novel feedback loops, then the molecular constituents of these systems would be gradually accumulated, thus increasing the likelihood of

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their survival. It is noted that in these simulations, the initial values used for parameters such as reactant concentrations and reaction rate constants may be higher than expected under ‘‘primordial’’ conditions, in order to demonstrate the development of feedback loops with their TSD reactions without using excessive computer time. On the other hand, it is recalled that during the dehydration period characterizing the fluctuating environment under consideration, very high concentrations may be achieved. The initial values of four short catalytic peptides were 109 M, the concentrations of each of the two amino acids under consideration were 106 M, the concentrations of mono-protonucleotides were 105 M and the concentrations of the relevant proto-tRNA were initially zero and after a relatively short time period of about 100 days reached 109 M. The loading reaction was the ratelimiting step in the development of a feedback loop with its TSD system. Recently, we explored some details related to the kinetics and extent of the loading reaction by starting from the analysis of the experimental results reported by Francklyn and Schimmel (1989), who studied the aminoacylation of tRNA and its minihelix (Yusim et al., 2001). In Nir and Lahav (1997), the overall loading was expressed as a three-body reaction: d½AR=dt ¼ kloading RAE;

ð4Þ

in which R, A, E and AR denote the concentrations of primordial t-RNA, amino acid, primordial peptide catalyst, and loaded amino acid, respectively. Typically, the calculations employed kloading ¼ 107 or 109 M2 s1, whereas the value deduced from the analysis of the results of Francklyn and Schimmel (1989) was 1010 M2 s1. Let us consider A in the range of 106–107 M, the concentration of catalyst (E) between 1010 and 1011 M, and the concentration of nucleotide bases between 106 and 108 M, and further reduce kloading to the range of 106–108 M2 s1. Within this range of values, the model calculations by Nir and Lahav (1997) give for the time required for exponential growth of TSD systems a range of 104–106 years. However, if we consider that during a cycle of dry conditions the concentrations of relevant molecules could be dramatically increased, then the concentrations of R, E and A employed in Nir and Lahav (1997) might be even underestimated. The estimation of catalytic peptide concentration in the fluctuating environment under study is as follows. Due to its solubility, glycine can reach high concentrations during the dehydration period. Based on several condensation experiments (Lahav et al., 1978; Suwannachot and Rode, 1999), the percentage of glycine undergoing condensation in experiments simulating fluctuating environments is in the order of 1% of the total glycine concentration. Assuming that catalytic peptides constitute 0.1– 1% of the peptides formed, and a total glycine concentration between 104 and 103 M, the estimated concentration of catalytic peptides is between 109 and 107 M, i.e., the same or one to two orders of magnitude above the value used by Nir and Lahav (1997). If we consider two or three orders of magnitude increase in A in Eq. (4), then overall, under dehydration conditions the loading rate would be 102–105 larger, and consequently the estimate of the time required for the first stage of the emergence of life may be reduced to 102–106 yr. This range should be compared with Lazcano and Miller’ (1994) estimate of 106 years needed for the emergence of cyanobacteria. The emergence time in the order of several tens of years is in the order of the time suggested by White and Raab (1983). Thus, once the exponential stage is obtained, the synthesis of the peptides under study is very fast, and the peptides can reach a high concentration within a short period of time.

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3.16. Implications The biogeochemical background on which the ETIF theory is based has hardly been addressed so far as a novel and experimentally testable paradigm. In view of the explanatory power of the ETIF theory, it may be of special interest to experimentalists, where the experimental program encompassing the emergence of attributes such as short catalytic peptides, bio-homochirality and synthesis of primordial ‘‘living’’ forms. 3.16.1. Novel methodologies for the study of short peptide catalysts The ETIF theory for the emergence of life is based on the assumption of the presence in the relevant prebiotic environment of short, organic catalytic molecules, most notably peptides. Because of the large numbers of different peptides even in molecular populations of relatively short molecules, the classical methodology of their study is inadequate. Thus, rather than one peptide at a time, the new technologies of libraries of molecules should be applied to the identification and selection of catalytic peptides in the model reactions under study. 3.16.2. Catalytic properties of short RNA molecules The size of the ribozymes studied so far is in the order of 100 or more bases and therefore cannot be considered ‘‘prebiotically plausible’’. A plausible prebiotic environment is characterized by features such as minerals, high ionic strength and wetting–drying cycles. A novel approach to the study of prebiotic ribozymes should thus take these features into considerations. Moreover, the recent proposal regarding the structural role of proteins in the active site of the peptidyl transfer mechanism catalyzed by RNA (Ban et al., 2000; Cech, 2000; Muth et al., 2000; Nissen et al., 2000), suggests a novel experimental approach of the study of primordial peptide bond formation involving both RNA and peptides. 3.16.3. Emergence of bio-homochirality The role of glycine in the emergence of bio-homochirality according to the ETIF theory can be experimentally simulated. In view of the experimental feasibility of peptide bond formation between glycine and alanine under prebiotically plausible conditions (see above), the emergence of homochirality in a test tube also seems to be a straightforward approach. 3.16.4. Emergence of a feedback loop in a test tube The scenario of the ETIF theory is characterized by an intrinsic rhythm that can easily be augmented in order to enhance the evolution rate of the chemical entities under study in the test tube, as discussed earlier (Lahav, 1985). The most fascinating possibility of this kind would be to explore the emergence time of a feedback loop with its TSD system, taking into account the presence of both minerals and short peptides as essential constituents of the prebiotic environment under study. In this regard, our computer model (Nir and Lahav, 1997) may serve as a first step in the planning of such an experiment. For instance, it is suggested that an exponential increase of the growth rate of the feedback loop or individual constituents thereof would serve as an indication for the emergence of a feedback loop (White and Raab, 1982). In order to facilitate the emergence time of a feedback loop, the wetting–drying (and/or heating–cooling) cycles of the simulated fluctuating environment can be greatly enhanced; in this way, an enhancement in the

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overall rate by several orders of magnitude seems possible, thus suggesting that the synthesis of primordial organizational units of living entities, i.e., feedback loop–TSD systems, is a rational goal of the search for the origin of life.

4. Concluding remarks According to the ETIF theory, the mechanism of biological and biochemical continuity along the entire evolutionary pathway leading from inanimate to animate matter and to extant forms of life is based on feedback loops and their template-and-sequence-directed (TSD) reactions. The implications of this theory encompass the emergence of the most fundamental attributes of life, i.e., the first feedback-loop systems with their TSD and catalyzed reactions, emergence and manipulation of information and bio-homochirality. This theory shows that the progress made by researchers of various disciplines suggests that certain evolutionary stages can be understood in relation to both top-down and bottom-up research strategies. This merging of the top-down and bottom-up viewpoints includes primordial short catalysts and templates as well as certain features of the emergence of the genetic code. Moreover, it provides a novel definition of life that serves as a common denominator to all living forms during the entire history of life. The experimental implications of the ETIF theory are far reaching, encompassing formation of feedback loops in a test tube and symmetry breaking in a racemic solutions of amino acids. Acknowledgements We are grateful to Ken Nealson for reviewing an advanced version of our manuscript. Discussions with D. Avnir, I.N. Berezovsky, A.M. Bergareche, M. Di Giulio, B. Green, R. Kasher, R. Lahav, A. Lazcano, K. Matsuno, S. Lifson, K. Nealson, S. Santoli and E. Trifonov are appreciated. We thank E.N. Trifonov and I.N. Berezovsky for providing us with their papers prior to publication, Ruth Eilon for help in various calculations word processing, and S. Zioni for the graphic work.

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