The First Cell

The First Cell Arthur L. Koch1 and Simon Silver2 1

2

Biology Department, Indiana University, Bloomington, IN 47405-6801, USA Department of Microbiology and Immunology, University of Illinois, Chicago, IL 60612-7344, USA

ABSTRACT The First Cell arose in the previously pre-biotic world with the coming together of several entities that gave a single vesicle the unique chance to carry out three essential and quite different life processes. These were: (a) to copy informational macromolecules, (b) to carry out specific catalytic functions, and (c) to couple energy from the environment into usable chemical forms. These would foster subsequent cellular evolution and metabolism. Each of these three essential processes probably originated and was lost many times prior to The First Cell, but only when these three occurred together was life jump-started and Darwinian evolution of organisms began. The replication of informational molecules that made only occasional mistakes allowed evolution to form all the basic components of cellular life. Ribozymes, the first informational molecules, were also catalytic. Energy coupling required the formation of a closed lipid surface to generate and maintain an ion-motive gradient. The closed vesicle partitioned components and avoided dilution within the primordial sea. Closed membranes were essential for the first selfreproducing cell to arise and for its descendants to disperse. Subsequent cellular development after the origin of The First Cell led to the beginnings of intermediary metabolism and membrane transport

ADVANCES IN MICROBIAL PHYSIOLOGY VOL. 50 Copyright r 2005 by Elsevier Ltd ISBN 0-12-027750-6 All rights of reproduction in any form reserved

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processes. This long process, subject to strong evolutionary selection, developed the cellular biology that is now shared by all extant organisms.

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. The Startup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. The Academy of the Origin of Life. . . . . . . . . . . . . . . . . . . . . . 2. Pre-biotic Chemiosmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Surfaces versus Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The Second Important Conclusion from the Miller–Urey Experiment . 4. Carbon in Biologically Useful Oxidation States . . . . . . . . . . . . . . . . 5. The Next Step Was the Generation of Biologically Important Small Organic Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Formation of Cell Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Uphill Energy Conversion and Ability to Drive Reactions . . . . . . . . . 8. The First Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. How to Make RNA Inside a Vesicle . . . . . . . . . . . . . . . . . . . . . . . . 10. Pre-Protein Polypeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Free Radicals and Ultraviolet Flux . . . . . . . . . . . . . . . . . . . . . . . . . 12. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. INTRODUCTION The goal here is to propose how the first ‘‘proto-cell’’, called here ‘‘The First Cell’’, arose on this planet. It is assumed (as is standard in recent booklength considerations of this question (Miller and Orgel, 1974; CairnsSmith, 1990; De Duve, 1996; Maynard Smith and Szathmary, 1999; Horneck and Baumstark-Khan, 2001) that cellular life arose de novo on Earth. The alternative, the arrival of The First Cell on this planet in a meteorite from another planet (i.e. panspermia, see Horneck and Baumstark-Khan, 2001), is not considered. In any case, cellular life would need to have arisen elsewhere from pre-cellular forms, leaving the basic problem unchanged (see Line, 2002). The pre-biotic steps preceding The First Cell, and then evolution from The First Cell to The Last Universal Ancestor and then to the current wide range of life forms (Woese, 1998) are outlined in Fig. 1. There is debate about whether a single Last Universal Ancestor (as we use the term here) existed as a single ‘‘species’’ or was more of a ‘‘Last Common Community’’. However, the task here is to explain the formation of The First Cell. Earlier and later stages were difficult for development of early life, but stages after

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Figure 1 The evolutionary tree of early life, stretching from abiotic processes that involve astrophysical and geological chemistry at the bottom of the figure to the three primary groups of diverse modern organisms at the top. The point here is the three abiotic processes (chemiosmosis, ribozymes, and vesicle formation) that combined within a single vesicle to form The First Cell.

The First Cell are easier to imagine than that single event when the functional First Cell arose in a pre-cellular world. Life could start only after Earth had cooled sufficiently for pre-biotic chemistry to take place to generate pools of small organic building-block compounds. The importance of the extra-terrestrial sources has been discussed (Bernstein et al., 1999). With cooling, life started relatively quickly in geological timescales (Orgel, 1998; Lazcano and Miller, 1999). Given the astrophysical and geological circumstances that provided conditions where synthesis of biochemically relevant pre-biotic organic compounds could occur, then pools of these compounds would have formed repeatedly in the

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Figure 2 The First Cell with a membrane vesicle bilayer, an autocatalytic ribozyme and a membrane potential generated by inorganic redox chemistry.

non-living world. Four systems were essential for cellular self-reproduction. These were (a) the pairing of single strands of nucleic acids with complementary single-stranded nucleic acids, (b) early ribozymes that have a catalytic function, (c) the formation of closed lipoidal barriers (ancestors of the cell membrane) in the form of vesicles, and (d) energy transduction to drive ion gradient formation (Fig. 2). Before the time of The First Cell, these advances happened separately but had a limited future since the entities were not capable of reproduction and building on previous success. The First Cell and its immediate descendants progressively evolved the full range of constituents of cellular life, as we now know it. The development of cell biology was clearly a tremendous task, possibly greater in scope than the subsequent development of the diverse organisms after the Last Universal Ancestor. The trunk of the evolutionary tree in Fig. 1 constituting the development of cell biology followed slowly after the formation of The First Cell.

1.1. The Startup The startup problem basically is that several processes needed to occur independently. Much has been said about the invention of heritability and catalytic ability (Gesteland et al., 1999) but these issues are not central to the

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thrust of this chapter. The importance of vesicles to enclose polynucleotides and polypeptides has been less extensively considered (Chen et al., 2004; Chen and Szostak, 2004; Hanczyc and Szostak, 2004) and will be discussed below together with the initial primitive energy source of the vesicle. The origin of The First Cell is a more daunting task to understand than is subsequent cellular evolution and appears to defy sensible explanations. Such a process is more imaginable if only a few processes were present in The First Cell, rather than the many functions common to all cells today. It follows that the progression from The First Cell to the Last Universal Ancestor involved invention of numerous processes. A large number of separate pre-cellular trials might have been sufficient to get a complex cell going de novo? The essentially simultaneous assembly of DNA, ribosomes, protein enzymes, and intermediary metabolism, which are characteristics of current living cells, seems highly unlikely to have been present in The First Cell (Fig. 2). It seems more likely that life arose with only a few processes and that The First Cell was importantly simpler than cells in existence at that time. What is a cell? A ribozyme on the surface of an iron pyrite crystal or a liposome membrane bilayer is not a cell but it is possible to suggest a series of stages by which either could evolve into a cell. A ribozyme within a lipid membrane (Chen et al., 2004; Chen and Szostak, 2004; Hanczyc and Szostak, 2004) seems a reasonable intermediate stage. We consider, however, the origin of The First Cell to be an actual event in time, when cellular life truly started: i.e., when all that was essential came together in a single vesicle. This was the generation of a cell that could grow and yield viable descendants that in turn could evolve. ‘‘Pre-cellular evolution’’ of chemical processes such as development of self-replicating ribozymes could lead to evolutionary selection but ribozymes by themselves could not move toward being a cell. The moment of origin of The First Cell is in a fundamental sense also the moment of the start of Darwinian organismal evolution. Once a vesicle developed mechanisms for maintaining useful concentration gradients (i.e., energy coupling) and enclosed a self-replicating ribozyme that could catalyze accurate nucleic acid synthesis (Johnston et al., 2001), then it was alive. All that followed was driven by evolutionary selection and rare errors (mutational changes) to form products functioning differently in small increments – Darwinism had started. Evolution leading to more complicated advanced cells requires (using far different words than Alfred Russel Wallace or Charles Darwin could have imagined) precise replication by catalytic function and occasional mutation. Innovative evolution allowed the relatively rapid (in geological timescale) development of the sophisticated and effective processes shared by all of

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today’s known cells. Transfer of catalytic function from RNA to proteins came after The First Cell. The First Cell did not contain protein enzymes. Ribosomes came later. The division of lineages at the Last Universal Ancestor stage with development of the extensive diversity of life forms (Woese, 1987, 1998) and eventual formation of eukaryotes by symbiotic fusion of Bacteria and Archaea followed considerably later (Fig. 1), perhaps 300 million years or more after The First Cell formed. All of the early processes diagrammed in Fig. 1 happened during the remarkably short time after the Earth’s surface cooled below 100 1C about 4.2–4 billion years ago. First, abiotic pre-cellular developments led to the origin of The First Cell perhaps 3.8 or 3.9 billion years ago, and then the broad diversity of microbes was in place by 3.5 billion years ago (Woese, 1998). Measurements about chemical conditions at the time of the origin of The First Cell come from the ratio of carbon isotopes in carbon samples of various ancient sedimentary materials (Strauss et al., 1992; Schidlowski, 2001). Carbon isotope fractionation occurred during the fixation of carbon dioxide and carbon monoxide, because the reactions favor the lighter isotope over the heavier, as happens today when cells carry out photosynthetic fixation of CO2. From the appearance of The First Cell onward, fractionation would have occurred. The carbon isotope fractionation of biologically-fixed carbon would have occurred well before oxygen-producing photosynthesis came into full swing with cyanobacteria, long after the time of the Last Universal Ancestor (Woese, 1987, 1998; Strauss et al., 1992). Similarly, evidence for microbial S-isotope fractionation has been found in rocks 3.5 billion years ago. Except for the creation of The First Cell, many of the processes just listed are amenable to current experimental study in the laboratory. In contrast, the problems associated with the transition of a pre-cellular vesicle to The First Cell are inaccessible both because it was a single event rather than a long process over time and also because of the lack of relevant available substrates and knowledge of the earlier stages.

1.2. The Academy of the Origin of Life Studies of the Origin of Life are quite old. For example, 2400 years ago, Aristotle speculated on this question. Modern efforts started with the monograph by Oparin in 1924 and then recent editions (Oparin, 1953, 1964). Miller in 1953 (see also Miller and Orgel, 1974; Miller, 1992) studied conditions for ‘‘pre-biotic biochemistry’’ and proposed involvement of purely physical and chemical processes in synthesis of precursor small organic substrates for current cells. These experiments and subsequent experimental

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work by many workers established reasonable conditions for pre-biotic synthesis of amino acids, nucleotides, and other small organic molecules. There are recent arguments contrasting a ‘‘warm dilute soup’’ as the precellular milieu with alternative hypotheses, such as whether The First Cell arose under conditions of higher temperature than most contemporary organisms can bear (geo-thermal, volcano-like) (Wa¨chtersha¨user, 2000). Recently, Brack (1998) and others reviewed these topics in depth, so we can skimp on this problem here. As to The Origin of The First Cell, the earlier dominant concept of the involvement of proteinaceous coacervates (Oparin, 1953, 1964) has fallen from serious consideration. There has been less exposition on the essential point made here that The First Cell was defined by having several simultaneous processes invented, rather than having The First Cell contain specific substrate molecules. A few quite unrelated but essential processes were needed and these needed to function together within the same proto-cell. Thus, the biochemical prologue to The First Cell has been actively addressed by experiments, analysis, and hypotheses over the last 75 years. During this time, a small group of interested experimental workers and theoretical modelers (‘‘The Academy of the Origin of Life’’) has functioned with, however, few generally accepted conclusions. Studies of The Origin of Life have not progressed along the normal path of the development of science with an iterative cycle of hypotheses followed by experimental tests. Rather, the problem has developed in a manner similar to that which Kuhn (1996) pointed out for other subject matter, such as early 20th century physics and mid-20th century molecular biology. Studies of The Origin of Life have consisted of a series of starts and stops, with conflicting paradigms shared by relatively small groups of workers. The ideas have been elaborated upon by proponents, but rather than being established by a succession of experiments, they fade away as the people involved leave the scene, to be replaced by younger workers whose thinking frequently does not incorporate much from previous generations. There has been much discussion over years concerning bioenergetics and the ‘‘high-energy’’ small molecule intermediates (usually assumed to be organophosphate compounds) used by early life. Was The First Cell heterotrophic (depending on pre-biotic organic substrates provided in the ‘‘primordial soup’’), chemically autotrophic, or even photosynthetic? Did it require a respiratory electron transport chain coupled with some external oxidant, as do most current life forms? With respect to the earliest bioenergetics, a reasonable guess is ‘‘none of the above’’. The just-listed processes are too complex and require support systems that are too sophisticated. As above, this question is not the major concern here, and relevant general reviews cited in the bibliography address these questions.

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The most likely pre-biotic source of the first usable energy for life processes is related to the possibilities proposed by Wa¨chtersha¨user (1988, 1990, 1994, 2000), who suggests that small organic pre-metabolites (and even polymers formed from them) were initially synthesized and stably maintained on the surface of insoluble iron pyrite particles, with energy derived from oxidation/ reduction reactions involving ferrous iron and H2S conversion into iron pyrites. These substrates were abundant in the anaerobic early world. We consider the mechanisms of Wa¨chtersha¨user (1988, 1990, 1994, 2000) as a partial alternative for generating the ‘‘primordial soup’’ to the irradiation processes of Miller (1992) (see Miller and Orgel, 1974) or from extraterrestrial fallout of meteorites (Deamer et al., 2002). The chemistry proposed by Wa¨chtersha¨user (1988, 1990, 1994, 2000) appears appropriate for making pre-metabolites including compounds containing several carbon atoms. However, the next step of energy coupling (Koch, 1985; Koch and Schmidt, 1991) must occur in a membrane vesicle, so that inorganic redox process could provide the basis for early pre-biotic chemiosmosis with the formation of a proton gradient. We consider the idea of Wa¨chtersha¨user (1988, 1990, 1994, 2000) of life originating on an inorganic solid surface implausible as proposed, since the surface redox chemistry on an inorganic crystal would not lead to the required chemiosmotic gradients across a lipid membrane to form a utilizable form of energy. There is no associated genetic ribozyme. New ideas and experimental work on the Origin of Life are quite intense today, driven by the ancient desire of humankind for an explanation of how we got here. There are other driving factors, such as the need of the National Aeronautics and Space Administration (NASA, USA) to be concerned with life elsewhere in the universe and the National Science Foundation (USA), and basic science agencies in Europe and in North America to understand the diversity and complexity of this world’s current biota. The extraterrestrial origin of life has been considered. For example, life might have previously existed on and come from Mars, embedded in meteors. The evidence of water below the current Martian surface and perhaps on Titan, a moon of the planet Saturn, provides the newest support for this possibility. One recent example with great press coverage was the claimed finding of ‘‘nannobacteria’’ in meteorites of Martian origin in Antarctica. It seems very unlikely that these structures are the fossil remains of primitive living Martian cells (Nealson, 1997). The question of the minimum size needed for a functional cell has been discussed (Koch, 1996; Szostak et al., 2001; Trevors and Psenner, 2001). The diameter of these nonliving meteorite structures is too small to maintain a potential gradient and to house informational and catalytic macromolecules, so these tubular structures are probably abiotic. (‘‘Nanobacteria’’ is also a term used for

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unusually small bacteria which occur abundantly under semi-starvation conditions. These nanobacteria are not involved in concepts of The Origin of Life – however, see the contrary recent view (Trevors and Psenner, 2001); note that the word is usually spelled with one ‘n’.) Lengthy reviews and monographs on The Origin of Life appear frequently, and these consider more fully alternative hypotheses and conjectures. Here, we are not attempting to pull diverse threads together in a contemporary summary. The primary point of this review is to emphasize that brief moment in time (over a second or a few million years) when it all came together and went from ‘‘pre-cell’’ to The First Cell. Whereas many studies have been concerned with the pre-biotic processes, fewer concern the stage between The First Cell and the Last Universal Ancestor (Fig. 1), during which time cell biology processes were invented and almost fully developed. Many studies concern evolution between the Last Universal Ancestor and present day life (Woese, 1987, 1998, 2000; Cavalier-Smith, 2002) as different lineages formed of the diversity of living forms that we now recognize. Prominent recent full monographs and reviews on The Origin of Life include the following: De Duve (1996); Horneck and BaumstarkKhan (2001); Maynard Smith and Szathmary (1999); Miller and Orgel (1974); Brack (1998); Deamer et al. (2002); Cavalier-Smith ( 2001, 2002); Deamer, (1997); Lahav et al.(2001) and Morowitz (1992).

2. PRE-BIOTIC CHEMIOSMOSIS An essential need for The First Cell was energy. Not just energy, but free energy that could be used to drive metabolic processes. The only way that we can imagine the spontaneous generation of usable energy without complicated machinery is a primitive form of chemiosmosis. Wa¨chtersha¨user’s (1988, 2000) proposed formation of reduced redox state carbon compounds would not generate a membrane potential, as there is no membrane postulated. Without an enclosed membrane, iron pyrites and H2 produced from H2S and Fe2+ do not lead to a usable supply of energy. Although inorganic redox reaction chemistry linked to carbon bond formation would be a form of pre-metabolism, a membrane vesicle is needed for pre-biotic chemiosmotic charge separation to occur and energy to be stored in The First Cell for use in cellular processes. Koch (1985) and Koch and Schmidt (1991) suggested that the elements of molecular H2, the other product of pyrite production, might react with CO or CO2 as proposed by Wa¨chtersha¨user (1988, 2000). However, Koch and

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Figure 3 A model for early energy coupling. Geologically available Fe2+ and H2S on the outside of a vesicle generated a chemiosmotic proton potential. The products are pyrite (FeS2), 2 H+, and 2e
. The cross-membrane potential forms when electrons enter the vesicle interior, transported via metal cations. Then CO or CO2 that has diffused to the inside can be reduced to formaldehyde and carbonate. This generates a proton potential that could be coupled to drive energy-requiring processes (adapted from Koch, 1985; Koch and Schmidt, 1991).

Schmidt (1991) proposed a different process producing a proton potential across a lipid membrane vesicle (Fig. 3). It requires that the protons remain on the outside of the vesicle where the iron pyrite forms and precipitates. The electrons move through the lipid layer associated with mobile metal cations and react inside, leaving the plus charge on the outside and the minus charge on the inside of the vesicle membrane. The electrons that enter the vesicle might react with CO or CO2, which can passively penetrate lipid membranes. The (charged) protons would be membrane-impermeable. But how could electrons enter the protocell? In modern cellular organisms, small organic molecules or protein carriers move electrons across membranes in respiration and in photosynthesis. It seems likely that in The First Cell (which lacked proteins as these are now understood) transition metal cations (Fig. 3) and/or sulfur compounds could have functioned in this role, although presumably more crudely than current electron carriers. Fe–S ‘‘cages’’ analogous to those found in ferrodoxins and redox chemistry similar to that carried out by ferrodoxin today are proposed here as central to early bioenergetics in the protocell. Such inorganic electron carriers and processes would be expected to predate membrane proteins in The First Cell. The range of known biological iron sulfur compounds has increased recently (Johnson, 1998) and details of how they function in

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oxidation/reduction reactions provide models for possible earlier processes that have been replaced by subsequent evolution. We suggest that a chemiosmotic process provides the reductant for The First Cell to fix CO2 and CO (Fig. 3). This was used for millions of years of cellular evolution, during which DNA, ribosomes, proteins, osmotically resistant cell walls, and enzyme-catalyzed intermediate metabolism evolved together with energy-generating processes. All of these subcellular components were present in the Last Universal Ancestor as long ago as 3.8 billion years (Orgel, 1998; Mojzsis et al., 1996). Only later did Bacteria and Archaea lineages separate and branch off rapidly into perhaps 20 current deeply rooted prokaryotic groups, only a few of which carry out photosynthesis (initially anoxygenic and later oxygenic). Methanogenesis, which also alters the isotopic carbon ratio in the biosphere, came after the first Archaea.

2.1. Surfaces versus Vesicles There remains the major question as to whether the earliest, pre-cellular macromolecules formed on solid surfaces, with clay-like templates (Wa¨chtersha¨user, 1988, 1990) or within membrane vesicles (Koch, 1985; Deamer, 1997). Pre-cellular accumulation of pools of small organic molecules may have occurred both within vesicles and by adsorption on clay-like surfaces. However, it seems likely that chemistry on unbounded open surfaces could not lead to the generation of a proton potential or to the formation of cellular descendants distributed through space and able to evolve in a Darwinian sense. Strong opinions about several topics (for example, chemical energy sources, pre-biotic organic synthesis, heterotrophic synthesis, and ‘‘warm soup’’ versus ‘‘hot soup’’) have attracted experimental modelers of the origin of life, but these questions are seemingly easier to evaluate in contrast to the large one emphasized here of the coincidence of the three improbable and unrelated processes needed for Darwinian evolution to begin.

3. THE SECOND IMPORTANT CONCLUSION FROM THE MILLER–UREY EXPERIMENT Although the major requirement for the creation of The First Cell was the development of an entity that could self-replicate, it is pertinent to consider the formation of pools of small organic molecules in an abiotic world. The

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physical location and long-term preservation of these pools in the earth is critical. In the classical experiments of Miller and Orgel (1974), electrical arc sparks and ultraviolet irradiation excited mixtures of small molecules in a gas phase. Later, special mixtures were irradiated (Ring and Miller, 1984; Miller, 1992). These physical processes supplied energy sufficient to form and to destroy chemical bonds. For example, the energy quanta in lightning are more powerful than those in carbon–carbon covalent bonds, and function in a non-selective way. With the simple gas mixtures that Miller used, molecules were formed that were somewhat larger than the starting gas molecules, but still not macromolecules. Of course, the newly formed organic compounds were also broken down. A key finding from these experiments is that some of the organic molecules (for example, amino acids) that were formed are components of current living organisms. However, in addition to finding abiotic pools of biochemical precursors, there was an insightful design aspect of the Miller apparatus that led to an appreciation of how a geological/meteorological process might function to generate and then to store organic molecules. In the apparatus used, water boiled and then condensed after exposure to radiation of the water vapor/gas mixture. This repetitive process enriched the aqueous phase with pools of organic molecules that were physically located away from the destructive irradiation. This occurred simply because they were less volatile than the original mixture of H2, NH3, CO2, and CH4. This flux of larger molecules (formed by irradiation) out of a liquid aqueous phase amounted to a ‘‘burying’’ or sequestering process in which the synthetic reaction products were trapped away from destruction by further exposure to the energy source that made them. Without the burying process, the irradiation would make and destroy the organic molecules, so that when steady-state equilibrium had been achieved, there would only be low levels of accumulated larger molecules. Given the specific gas mixture, temperature, pressure, and other relevant thermodynamic parameters, the equilibrium concentrations can be calculated and are very small. On the other hand, the burying process would generate much higher concentrations of more complex molecules than would simple thermodynamic equilibrium, even in the absence of any selectivity of the energy source (Fig. 4A). In the Miller experiments, the burying resulted from solution of the generated glycols, amino acids, heterocyclic compounds, and carboxylic acids in water remote from the energy source. Burying also could occur by absorption on to soil or clay surfaces, which then became covered with other materials. The original small organic products of the geological or meteorological processes were eventually physically covered and separated

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Figure 4 (A) Accumulation by burying versus production and destruction by irradiation. Only low-product concentrations could persist in an environment where a substance is made and simultaneously degraded by an energy flux. The units on both scales are arbitrary, as this figure is from a general modeling that allows wide ranges depending on rates of formation and degradation, and the rate of burying and extent of synthesis and burying. Burying over a fourfold range is shown, increasing accumulation at a site protected from the irradiation flux. (B) Non-destruction conversion through a sequence of organic compounds. Sample kinetics are shown for a model in which the input molecule C1 is converted into C2, then C2 molecules are converted into C3, etc. Eventually, a considerable fraction is converted into the much-changed product C4.

from energy sources by dust, volcanic ash, tidal flows, plate tectonic overturns, and other large-scale processes. Then later, the pools of organic compounds were re-exposed and polymerized to larger and eventually functional macromolecules. It was recently modeled (Mulkidjanian et al., 2003) that protection of phosphodiester linkages by UV-absorbing nucleotide rings was an early pre-cellular evolutionary pressure favoring longer and more complex RNA-like polynucleotides. The kinetics of pool-generating processes is reminiscent of (but not functionally related to) enzyme kinetics in that an intermediate is formed and then converted with some probability into the reaction product. The kinetics of these geochemical processes therefore might follow (analogous to the Briggs–Haldane version of Michaelis–Menten two-step reaction mechanism for enzyme kinetics) the following form: Ek1

k3

S1 þ S 2 # S 1;2 ! Products Ek2

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This leads to v ¼ ½S1;2 k3 ¼ ½S 1 ½S 2 k1 k3 =k2 , where v is the velocity of the overall process and S1 and S2 the concentrations of the two initial reactants. Ek1 and Ek2 are the forward and reverse rate constants for the initial chemistry in the presence of the geophysical energy flux, E. S1,2 is the activated intermediate product and ‘‘Products’’ is the final mixture that is buried in a unit of time; k3 is the rate constant of the one-way burying process. The accumulation of more complex molecules occurs even where there is no selectivity to the action of the energy flux. Note that the energy flux drops out of the equation and also that there is no hyperbolic (saturable) dependence on substrate concentration, because there is no fixed number of catalytic centers, as in enzyme kinetics. The integral of v over geological times would lead to the accumulation of considerable amounts of product, as shown in Fig. 4A. In contrast, the concentration of an intermediate or product substance in the environment without burying would be the equilibrium between synthesis and its degradation by radiation, which is represented by the horizontal line shown at the bottom of the graph. Accumulation shown by the various curved lines in Fig. 4A span a fourfold variation in the rate of burying and protection. This accumulation would occur even if the equilibrium constant, k2/k1, for the coupling step were small, or if the rate constant for burying were small and fluctuated with circumstances from time to time. The return of the buried material into the hydrosphere after physical removal from the ‘‘dilute soup’’ would depend on geological processes.

4. CARBON IN BIOLOGICALLY USEFUL OXIDATION STATES Carbon in its most oxidized (CO2) and most reduced (CH4) states is not a versatile building block for organic chemistry. Carbon at the intermediate levels of formic acid, formaldehyde, and methanol can be incorporated more easily into different and more complicated molecules. This is mainly because hydroxyl groups can be more readily exchanged for other carbon atoms or for nitrogen, sulfur, or phosphorus. If the most readily imaginable environments on early Earth came to thermodynamic equilibrium, carbon would end up mainly as either CH4 or CO2. Which alternative redox state would dominate depended on the abundance of reducing compounds, such

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as H2, or oxidizing compounds, such as SO2
4 or O2 in the environment. This in turn is a geophysical question. There are a few imaginable early environments where there would be stable equilibrium with intermediate oxidation states of carbon. One would be an environment with both CO2 and CH4. Such an environment might arise by geological mixture of material from different depths, for example, through volcanic action, with oxidized rock containing, for example, silicates exposed to a more reducing atmosphere. Then appreciable levels of HCHO or its hydrates and polymerized forms would be subject to the Miller scenario and then buried. This leads to a situation that can be modeled very simply: C1 ! C2 ! C3 ! C4 !    ! Cn . Sample kinetics are shown in the successively numbered curves in Fig. 4B. Consideration of whether there was too much or too little H2 available on the surface of Earth for the origin of life is critical. For the origin of The First Cell, there were two constraints. The first was the need to favor the formation of lipid vesicles. For this, the formation of aliphatic hydrocarbons and later of carbon chains with hydrophilic functional terminal groups was required. Only after The First Cell formed would the equivalent of glycerollipids – and eventually phospholipids – arise. When thermodynamically long times are under consideration, it was important not to have too much hydrogen or the available carbon would form methane and be lost to the atmosphere. In carbonaceous meteorites, there are few hydrocarbons while there are substantial amounts of polycarbon compounds. This is expected since meteorites pass through high-vacuum space and any CH4 or H2 would be lost by evaporation. The main carbon constituents of meteorites are cyclic carbon compounds (Deamer et al., 2002) with low hydrogen content and low volatility. Thus, for the generation of hydrocarbons on early Earth, more hydrogen (and atmospheric pressure) was necessary (Brack, 1998). Subsequently, fatty acids (as found today in Bacteria and Eukaryotes) and polyalcohols (as found now in Archaea) could arise. With turbulent mechanical stirring of a liquid water phase, the lipids would form membrane vesicles. Physical forces might have limited the formation of mixed vesicles contained in both types of lipids, with resultant separate fatty ether and fatty acid vesicles. This possible abiotic separation process into fatty acid vesicles and ether-linked lipid vesicles is a hypothesis that could be subject today to direct experimental test. Evidently, a variety of lipids formed membrane vesicles, and standardizing them was a later event, at the time when still undifferentiated prokaryotes split off to form the Bacteria and the Archaea.

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To form useful pools of pre-cellular pre-metabolites, such as amino acids, nucleic acid bases, and pentoses, a lower level of H2 would be required than that associated with pre-cellular lipid hydrocarbon synthesis. One might conclude that widespread environmental switching between high- and lowambient hydrogen levels was probably required in the development of precellular pools prior to The First Cell. Out-gassing from deep sediments formed the earliest atmosphere and increased the partial pressure of H2. Prelipids and more oxidized pre-metabolites might have arisen at different times and/or in physically separate locations, later to be mixed together by largescale geophysical upheavals. Only then were pools of amino acids, heterocyclic bases, and nucleotides formed and mixed together. Evidently, an alternating two-stage variable-environment version of the Miller experiment should be carried out.

5. THE NEXT STEP WAS THE GENERATION OF BIOLOGICALLY IMPORTANT SMALL ORGANIC MOLECULES The range of sizes of primary substrates involved in cellular intermediary metabolism is quite small, most having 5–25 atoms of C, H, O, P, N, and S. The burying process would not involve just two substrate species S1 and S2, as in the model above, but many different products would have been accumulated. A wide range of small molecules, with varied functional groups that subsequently served as the basic building blocks of pre-living complexes were generated and retained abiotically. These had various proportions of carbon, oxygen, hydrogen, nitrogen, phosphorus, and sulfur and were fundamental components of the pre-biotic world. Along the path to the generation of The First Cell, physical forces and chemical bonds that depended on the special properties of water were crucial. While cellular macromolecules are made of small organic precursor molecules that are connected by covalent bonds, the assembled precursors are later held together both by covalent bonds with oxygen or nitrogen and by additional bonds (ionic, H-bonds and hydrophobic Van der Waals forces), weaker and quite different from the covalent bonds studied in the Miller experiments. For example, van der Waals and other apolar forces hold the phospholipid molecules in membrane bilayers together. These lower-energy bonds can be formed and broken under conditions that do not destroy the covalently connected organic precursors of macromolecules.

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6. FORMATION OF CELL MEMBRANE Several sequential stages are recognized. There were long insoluble hydrocarbon chains synthesized by non-biological means in the primordial soup (Deamer et al., 2002). Oxidation of the terminal ends of the hydrocarbons created amphiphiles. Wave action and bubble formation would bring about the formation of liposomes, i.e., small spherical vesicles bounded by amphipathic lipoid layers with the hydrophilic end groups covering the hydrocarbon interior of the membrane. The intra- and extra-vesicular aqueous environments were separated by the hydrocarbon layer. For chemiosmotic energy processes, the layer served as an electrical insulator and physical barrier. All bacteria and eukaryotes exclusively use fatty acids linked by ester bonds to a glycerol moiety (Fig. 5A) in their lipid bilayer structures. Covalent addition of ethanolamine and charged residues that create phospholipids probably came later. The lipids in Archaeal membranes lack ester linkages, but have instead long hydrocarbon chains (including isoprenoids)

Figure 5 Cellular hydrocarbon diversity. (A) Bacterial lipids with ester linkages and an example of a phospholipid. (B) Archaeal lipids with ether linkages and examples of di- and tetraether lipids.

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linked together by ether linkages (Fig. 5B). How did these differences arise and then how did two forms both survive early Darwinian competitive selection? Again, this can be phrased as a ‘‘which came first, the chicken or the egg’’ question. Without doubt, both Bacteria and Archaea descended from the same First Cell that used mixtures of lipoid molecules produced pre-biotically for the membranes of The First Cell and did not develop pathways for biosynthesis of different lipids until much later. Probably, The First Cell depended on abiotically synthesized hydrocarbon lipids, which lacked both ester and ether linkages. These came later as did DNA, RNA, ribosomes, and most of central metabolism that are shared without major differences between modern Bacteria and Archaea. One cannot solidly conclude today as to which came first during development of metabolism after The First Cell, Bacteria- or Archaea-like lipids. If only three biological processes were required in The First Cell but there were hundreds of enzymatic steps and macromolecular biosynthetic steps that functioned in the cells of the Last Universal Ancestor, most cellular evolution occurred between these two early stages of life (Fig. 1). The classes of cellular processes that evolved can be arranged in eight groups: (a) biosynthetic pathways for synthesis of metabolic intermediates, (b) polymerization for DNA, RNA, and protein synthesis, (c) use and storage of chemical and physical energy sources including chemiosmosis and ATP, (d) membrane-carrier proteins for transport of small molecules into and out from the cell, (e) determination of cell shape and reliable cell division, (f) sensors and signaling proteins for responses to environmental disturbances, (g) regulation of the cellular processes at the gene level and post-gene level and (h) nucleic acid repair processes. There is an open question of whether the stages between The First Cell and the Last Universal Ancestor all occurred in a single cell lineage (with the rapid replacement of early ‘‘experiments’’ with later more successful cells) or whether the intermediate stages shown in Fig. 1 occurred in a dynamic population of early cells, ‘‘The Last Common Community’’, or a ‘‘superspecies’’ (Woese, 1987, 1998, 2000, 2002; Boucher and Doolittle, 2000), rapidly evolving by selection and rapidly exchanging genetic information. Electrical discharges on the early Earth might have driven a primitive form of electroporation (a contemporary laboratory means of introducing informational RNA and DNA into cells by high-voltage electric discharges). Whether in a single species (in a modern Darwinian sense) or as a community of frequently gene-exchanging early cells, evolution had to take place step by step, by cells that worked better at each stage supplanting previous varieties. Then additional changes and possibly quite different processes would replace the former, early cells in series. Both before and after The

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First Cell, turbulence from massive storms would have caused the formation, breaking, and fusion of lipid vesicles with the result that, after The First Cell, sometimes independently evolving early cells would have found themselves sharing a single membrane and subsequent evolutionary history. Some authors assume (even when this is not clearly stated) that evolution took place simultaneously in many early cell lineages and that the advances were then funneled together by membrane fusion and/or by lateral gene transfer into a common descendant, eventually called the Last Universal Ancestor. Lazcano and Miller (1994) conclude from the geological and biological evidence that there were only 300 million years between the time that the surface temperature cooled enough for life to survive and the time of the development of cyanobacterial stromatolites (the oldest recognized fossils of living cells) perhaps about 3.8 billion years ago. Closely related questions are how long did it take for life to begin pre-biotic stages to (a) The First Cell, (b) then develop all of the cellular components of the Last Universal Ancestor cell, and then (c) establish diversity to the approximately 30 deeply rooted prokaryote (Bacteria and Archaea) lineages now found (Cavalier-Smith, 2002), and finally (d) to form the first eukaryote by symbiotic fusion of bacterial and archaeal prokaryotes? These are important, but beyond our scope of consideration. One early stage (but slightly after the Last Universal Ancestor) is the origin of Archaea. It was previously thought that Archaea arose before Bacteria, but it is now more generally considered that both groups of current prokaryotes arose from a no-longer existent, but shared, ancestor. It is sometimes suggested that the ether linkages of Archaea were a subsequent adaptation to the high temperatures and extremes of pH and salt characteristic of environments where many current Archaea are found. However, Archaea have recently been found in more moderate environments, such as the open ocean. Many aspects of their structure (cell walls, although of different structures) and metabolism (many shared cofactors, but also unique cofactors, for example, those required for methanogenesis) are quite similar for Bacteria and Archaea, making it very probable that both lineages separated long after the formation of The First Cell and evolution of widely shared cellular metabolism, most likely closer to the time of the Last Universal Ancestor cell. The initial vesicle that became alive would have enclosed some of the primordial ‘‘soup’’, although with little selection other than providing counter ions for membrane molecular head groups. It enclosed a nutritious, rather dilute, representation of the ancient pre-biological ocean. Of course, the amount of enclosed soup could not support significant growth without additional resources being continuously commandeered (that is, net uptake

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across membranes was an early required step). The osmotic pressure resulting from a ribozyme entrapped in a membrane vesicle results in that vesicle accumulating fatty acids at the expense of other vesicles and therefore growing in size (Chen et al., 2004). This result provides an interesting model for development within a pre-cellular vesicle, which then would need to be coupled with energy-dependent uptake of nucleotide-like precursors, for the development of very early cellular life. There are additional questions for consideration in the formation of the membrane of The First Cell. The first is that vesicles made by physical processes (such as sonication in the laboratory or foam formation by ocean storms) are generally too small in diameter to enclose a modern prokaryote cell. Although small proteoliposomes can generate a chemiosmotic membrane potential and use this to generate ATP (Racker and Stoeckenius, 1974; Deamer, 1997) and substrate gradients (Kaback, 1986; Abramson et al., 2004), these are energy-consuming processes in proteoliposomes. They do not result in usable long-term energy storing, as needed for function as a cell. Lipids in a hydrophilic environment can form a variety of structures. Phospholipids form bilayers (Fig. 6). As Tanford (1991) pointed out, this is favored when there are two aliphatic chains per charged head group. Hargreaves and Deamer (1978) showed that a mixture of long-chain fatty acids and alcohols could form bilayers because they aggregated to be similar in outer appearance to two-tailed phospholipids. The physical requirement is that the molecules should have their aliphatic tails pointed away from water and their charged (or hydrophilic) groups in the aqueous environment. Therefore, phospholipids assemble to form bilayers that spontaneously connect all ends to form closed vesicles. Closure is due to the physical forces that reduce the contact of the alkane side chains with the aqueous milieu that would exist at the edges of flat bilayers. It is important for the development of cellular life that vesicles with closed hydrophobic barriers develop, so that these can maintain a voltage gradient across the membrane, a vital requirement for The First Cell. Lipid-like bilayer vesicles would have occurred due to purely physical forces as soon as there were bodies of water mixed with heterogeneous hydrophobic compounds. Also spontaneously, as pointed out above, these vesicles would generate and maintain a transmembrane voltage in the right chemical environment. Vesicles and energy conservation, however, would not lead to a living cell unless the other requirements for Darwinian evolution were satisfied. There is differing pressure between evolutionary forces favoring diversity and those favoring uniformity. On the one hand, if massive lateral gene transfer occurred at an early time during evolution of cellular metabolism,

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Figure 6 (A) Bacterial membrane bilayer with fatty acids and embedded proteins. (B) Archaeal cell membrane with a monolayer of tetraethers and embedded proteins.

after the formation of The First Cell, and was reduced to a trickle shortly after the split at the Last Universal Ancestor stage, different kinds of organisms would be found only after exchange was reduced (almost by definition). There would have been a large range of transient environmental diversity while shared cellular physiology was developing. This has sometimes been referred to as a ‘‘Last Common Community’’. Differing variants would develop under Darwinian selection and the time between the Last Universal Ancestor and the major groups representing currently found microbial diversity might well have been brief in geological terms (Lazcano and Miller, 1994) . The early organisms that formed the three domains of current life after the Last Universal Ancestor were very different from one another in superficial aspects, while more basic attributes (such as central metabolism, ATP, DNA, and ribosomes) are quite similar. Maintenance of uniformity within a gene-exchanging species is a consequence of what ecologists refer to as the ‘‘Competitive Exclusion Principle’’, which was clearly part of Darwin’s thinking 150 years ago, before knowledge of genes and nucleic acids. The idea in modern terms is that, in a constant habitat, the fittest will survive and eliminate the less fit and yield only one species per specific environment. Mutations occur at the nucleic

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acid level and more successful cells will replace previously dominant varieties. Different varieties are selected in slightly different environments, but when brought into a single environment, only one will survive. This is an essential basis for arguing for a single ‘‘species’’ for the population of organisms in Fig. 1 that constituted the descendants of The First Cell and the predecessors of the Last Universal Ancestor. Thus, we favor a single First Cell (Fig. 1) lineage and numerous intermediates between The First Cell and the Last Universal Ancestor.

7. UPHILL ENERGY CONVERSION AND ABILITY TO DRIVE REACTIONS The chemiosmotic proton potential (Mitchell, 1968, 1979; Harold, 1986) probably arose abiotically (Wa¨chtersha¨user, 2000; Koch, 1985; Koch and Schmidt, 1991) and predated subsequent biological conversion of energy into ‘‘high-energy phosphate bond’’ compounds, such as ATP. An energystoring membrane chemiosmotic gradient was essential for the First Cell. High-energy phosphate bonds would have been generated early from prebiotic processes with phosphate-anhydride compounds formed by heating or irradiation, but this probably would not lead to a process of evolution, until replicating ribozymes formed. Later, evolution linked chemiosmotic and phosphate bond energy in a major step toward intermediary metabolism, generating a range of building block substrates inside early cells and leading to more efficient nucleic acid replication. The strong selective benefit of producing high-energy metabolic intermediates for nucleic acid synthesis within early cells freed them from dependence on the pre-biotic soup and was clearly a high priority. An interesting idea has been suggested (Hud and Anet, 2000) concerning RNA synthesis on an organic platform, possibly a derivative of phthalocyanine. In contrast, many students of the Origin of Life postulate development of extensive metabolism and energy coupling before the existence of nucleic acids (i.e. metabolism before heredity Wa¨chtersha¨user, 1988, 2000; Morowitz, 1992). We do not consider this possibility plausible and propose that nucleic acids, very similar to their current form based on five-carbon sugar polymers with information-housing complementary side groups, (Mulkidjanian et al., 2003) must have been present in The First Cell. Woese (2002) addressed the question of the origin of The First Cell recently with a new direction from his previous emphasis on 16S ribosomal RNA sequences and on relationships between existing living groups at the

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time of the Last Universal Ancestor. The First Cell had RNA and lacked DNA and modern proteins. Early peptides that were random and not encoded by nucleic acid may have existed (Woese, 2002) as we also argue (Fig. 2). Membranes are assumed with a need for energy coupling and compartmentalization, but never mentioned. Woese (2002) then proposes RNA translation to polypeptide sequences would evolve. He argues that there were no ‘‘species’’ at this time in a Darwinian sense and that evolution of species had not begun. If we turn this around and propose that modern evolutionary thinking supports a single species of The First Cell that would have quickly displaced less successful natural experiments, once RNAencoded polypeptide sequences appeared, evolution would favor both a rapid increase in accuracy of the translation process (leading to the ribosome with ribozyme catalysis of peptide bond formation) and then the transfer of catalytic roles to more efficient proteins. An early pre-cellular step in maintaining and transmitting information was the invention of complementary RNA sequence and base pairing along an RNA template, much as we recognize today. The RNA molecule with intrastrand base pairing possibly formed pre-biotically to protect against radiation damage (Mulkidjanian et al., 2003) and to catalytically cut and splice (Fig. 7). This autocatalysis was the first ‘‘bio-catalysis’’, even if precellular and not a robust process. There is some inconsistency in arguing that Darwinian selection required encapsulation (Fig. 2) and also that a form of pre-cellular evolution occurred with naked pre-cellular ribozymes. Encapsulation instantly made the playing field unleveled and justifies the concept of The First Cell (Fig. 2).

8. THE FIRST NUCLEIC ACIDS It is generally accepted that RNA predated DNA in pre-cellular and early cellular evolution. ‘‘The RNA World’’ (Gesteland et al., 1999) is the definitive statement of this hypothesis. A recent and concise review is available (Puerta-Ferna´ndez et al., 2003). Aspects of early pre-biotic and post-cellular development of ribozymes include the first self-replicating nucleic acid and subsequent stages, such as the development of the ribosome as the platform for RNA-encoded polypeptide synthesis (Gesteland et al., 1999; Doherty and Doudna, 2000) Newer publications on the RNA World include Doherty and Doudna (2000), Curtis and Bartel (2001), Doudna and Szostak (1989), Levy and Ellington (2001), Lohse and Szostak (1996), Salehi-Ashtiani and Szostak (2001), and Tang and Breaker (2000).

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Figure 7 (A) Self-splicing (Group II intron) and (B) trans-cleaving (hammerhead) ribozymes. (A) The steps are (1) An intron-internal adenosine nucleophilically attacks at the 50 end of the intron, releasing the 50 upstream exon 1 with a free 30 OH. (2) The 20 OH of the A forms an unusual 50 –20 phosphodiester bond to the conserved terminal 50 AUG of the intron, creating a ‘‘lariat’’ structure. Horneck and Baumstark-Khan, (2001) The 30 OH of exon 1 (still hydrogen-bonded to the intron) nucleophilically attacks the 30 end of the intron, after a conserved AG dinucleotide, forming the ligated exon1-2 and releasing the lariat intron with a 30 AG . (B) Hammerhead ribozyme hydrogen-bonded to substrate RNA that is cleaved and subsequently released. No ligation occurs.

More than 12 classes of ribozymes (catalytic RNA) are recognized (Tang and Breaker, 2000). Some function intrastrand (e.g., intron-removing activities) while many modern ribozymes cleave interstrand (e.g., hammerhead ribozymes and RNAse P processing pre-tRNAs to mature tRNA). RNAse P contains 10% polypeptide as well as about 90% RNA by mass, but it is the RNA that is catalytic (Gopalan et al., 2002) with the polypeptide increasing the efficiency (i.e., increasing the kcat). Additional ribozyme activities include the removal or addition of single nucleotides to paired 30 ends and the ligation of the two fragments after intron removal.

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Fig. 7A shows diagrammatically the reaction steps for one modern ribozyme that removes the intron and ligates exons. The more simple transcleavage by a ‘‘hammerhead’’ ribozyme involves extensive base pairing (Fig. 7B) as does the process in Fig. 7A. The complex is formed by a ribozyme polynucleotide with a substrate RNA that is subsequently cleaved. The hammerhead ribozyme can act repeatedly on new substrate RNA polynucleotides and this is true catalysis. Modern hammerhead ribozymes can contain both ribo- and deoxyribo-nucleotides ligated together in a single strand. In addition to cleavage and ligation, some ribozymes carry out polymerization and thus function as ‘‘replicases’’ (Gesteland et al., 1999). An important characteristic of ribozymes is the requirement of secondary and tertiary structure for activity (Doherty and Doudna, 2000). These include the stem-loops anticipating double-stranded DNA, and forming complex and compact tertiary-folded structures (Puerta-Ferna´ndez et al., 2003). In these branched molecules, single-stranded regions pouch out from double-stranded regions and then return to bind complementary sequence (Fig. 7B), giving these RNA molecules well-defined secondary and tertiary conformation, similar to current day tRNAs and ribosomal RNA. Considering tRNA with a single covalently attached amino acid as a model for the first ribopeptide, one can envisage building a polypeptide chain attached to the informational sequence of the RNA molecule, by a process analogous to current protein synthesis, but at a time predating ribosomes. The RNA and protein components would subsequently separate and no longer be attached covalently. However, the RNA would retain catalytic function and still later the protein sequence would assume catalytic activity (as in most currently known enzymes). These stages would all occur along the trunk in Fig. 1, after The First Cell. The division of labor continued, but informational and replication functions were retained by the RNA (and only later transferred to DNA). Most catalytic functions moved to the polypeptide portion of the molecule. When an RNA sequence came to encode a particular amino acid (i.e. to specify attachment to itself), the early genetic code began, probably with less than 10 codons at an early stage in the ribosome-free early cell (Koch, 1996). This code had far fewer than the universal 20 amino acids found in proteins today and the 64-codon universal triplet code evolved subsequent to the appearance of The First Cell (Fig. 1). However, it is likely that the code was always non-overlapping triplet and never involved two nucleotide codes. Since all living cells today share the same set of amino acids and basically the same genetic code, the 64-triplet genetic code was complete (Cavalier-Smith, 2001)

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and universal at some time before the time of the Last Universal Ancestor. Probably, tryptophan was the last amino acid to be added to the code. Selenocysteine came still later, as did the degenerate loss of the third stop codon in mycoplasma. Secondary amines, glutamine, and asparagine came after glutamic acid and aspartic acid; the early code probably did not distinguish between the two current dicarboxylic acids. Catalytic RNA was present in The First Cell, but polypeptide proteins had not yet been invented. This is an important conclusion from analysis of the minimum number of components needed for The First Cell, especially given the more usual emphasis in the literature on pre-biotic synthesis of metabolites, such as amino acids. For example, Wa¨chtersha¨user (1988, 1990) adheres to a scenario where metabolism predates information-encoding polynucleotides. We argue the converse. It seems likely that The First Cell contained within its membrane only RNA, and possibly a few catalytic peptides, and predated the invention of DNA. It has been argued that the take-over of the genetic encoding role by DNA resulted from the early invention of repair, by early protein enzymes that corrected replication mistakes in double-stranded DNA. The result was that all living cells then and now have double-stranded DNA encoding triplet codon heredity, and RNA now functions mainly as an intermediate between the DNA and the ribonucleoprotein-based protein biosynthetic machinery. Additional remnants of these early RNA-only cells have disappeared. Viruses contain either RNA or DNA, and are either single- or double-stranded ones. Viruses are more likely to have arisen from cells by degenerative reduction in function and are not thought to represent early preliminary stages toward more complex forms (and therefore are missing from Fig. 1). It should be emphasized that viruses and plasmids do not exist without living cells to parasitize.

9. HOW TO MAKE RNA INSIDE A VESICLE The problem of forming a highly charged macromolecule inside an early vesicle, rather than outside in the dilute soup, is a serious obstacle to the proposal that the origin of life took place inside a vesicle. However, ribozymes can be encapsulated in membrane vesicles and then form an abiotic membrane potential and drive growth of vesicle size (Chen et al., 2004; Chen and Szostak, 2004). For ribozymes to replicate inside vesicles, small, charged precursor molecules, such as phosphate-containing nucleotides, need to get across membranes and build up concentration gradients. It is possible that

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the early vesicle bilayers that became The First Cell were somewhat more permeable than current natural vesicles because of heterogeneous composition (as discussed above). An early non-protein-based form of RNA uptake similar to bacterial transformation might have been much slower than subsequently evolved protein-catalyzed transfer processes. Pinocytosis and phagocytosis are complicated processes and cannot be imagined to have occurred very early on; these are found only in contemporary Eukaryotes. A suggestion here is the possibility that early uptake of phosphate and phosphorylated compounds depended on the proton potential and on the surface charges of the membrane bilayers. The bilayer of a modern cell has negative charges on both faces, neutralized by magnesium cations (see Koch, 1986). This is a situation rather like the Debye–Hu¨ckel phenomenon of a ‘‘counter-ion atmosphere’’ of opposite charges surrounding a charged macromolecule and is considered in standard biochemistry textbooks. The counter-ion cloud would screen anionic phosphate and nucleotides from a negatively charged vesicle surface. If phospholipids were not the major element of the bilayers, that simplifies the problem for early life somewhat. The surface charge must tend to block nucleotide uptake (effectively a point charge approaching a charged surface), since a charged planar membrane will develop a charge potential extending typically for a few nanometers into the medium. The development of a proton potential (membrane external surface acidic and positively charged) favored the entry of negatively charged species, such as nucleotides and ribozymes. This means that given the development of a proton potential (Fig. 3), it would lower the pH in nearest nanometer to the surface by several units below that of the medium fluid (Koch, 1986). A consequence of this is that, on the outer face of the vesicle, the acidity is sufficient to protonate phosphate compound, reducing its net charge. Protonation would render phosphate compounds less lipophobic, and these might migrate across to the inner vesicle surface more readily, perhaps associated with non-specific amphipathic peptides (Fig. 2). In the nanometer immediately inside the inner surface, proton depletion could result in a functional pH higher than the immediate outside. Protons picked up on the outside would dissociate on the inside, with the pH gradient driving uptake of RNA precursors and short RNA chains. It is possible that such an early chemiosmotic potential might also favor phospho-ester bond formation. Overall, the mechanism would favor entry into the vesicle of phosphate and small-phosphorylated compounds. The process would be slow compared with those of current cells, but might be effective over pre-cellular and early cellular timescales.

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10. PRE-PROTEIN POLYPEPTIDES The first catalytic protein polypeptides were probably the peptide extensions from RNA ribozymes that provided better catalytic properties for these hybrid molecules or random peptides synthesized abiotically (Miller and Orgel, 1974). Those associated with the vesicle membrane or the ribozyme by non-covalent chemical bonds would not be subject to Darwinian evolution. Only those covalently attached to the ribozyme would evolve advanced catalytic function. Figure 8 Shows shows the structures of modern representatives of two possible classes of non-protein membrane peptides, gramicidin, and valinomycin. Both are made today by enzymes and not on ribosomes; both contain covalent linkages not allowed by ribosomal protein synthesis. They are synthesized by microbes, residue by residue, on large protein templates, which could not have existed in The First Cell or its immediate descendants. Nevertheless, they provide models for early peptides. Gramicidin A is a 15-amino acid long linear peptide, with alternating L- and D-amino acid residues (Fig. 8A), unlike modern proteins that contain only L-amino acids.

Figure 8 Cation-conducting membrane-soluble peptides (A) gramicidin, a linear peptide and (B) valinomycin, a cyclic dipsipeptide [http://pubchem.ncbi.nlm.nih.gov/ substance/].

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Gramicidin is incorporated into lipid bilayers, forming a narrow channel across the membrane through which inorganic monovalent cations can pass. Valinomycin is cyclic and contains 12 residues, i.e., six amino acids and six hydroxycarboxylic acids. Again, D- and L-acid units are found in valinomycin. Peptide and ester bonds alternate in valinomycin (Fig. 8B), which functions as a mobile carrier that picks up K+ cations specifically (but not Na+) at one membrane surface and releases the cation at the other surface. Thus, given a membrane potential (Fig. 3), an early valinomycin-like peptide could produce a K+ gradient (high inside/low outside) in The First Cell, similar to K+ gradients now a common currency of energy conservation in all cells.

11. FREE RADICALS AND ULTRAVIOLET FLUX Radiation was damaging to pre-biotic molecules and also to The First Cell. The free energy holding two carbon atoms together (C–C, and also C–S and C–N bonds) in covalent linkage is no more than several hundred kcal per mol. This is small compared with a quantum of cosmic gamma radiation. Free radicals can be generated and then disrupt organic molecules. In the early anaerobic world, oxygen free radicals were not as important as they are today. However, in a world where life was fragile and not as robust, how could life survive? Fundamentally, the answer is the same as that given above in discussion of the Miller experiment: life, as well as its small metabolite precursors, must have been buried for protection. It is clear that bacterial life exists today deep underground. This happens when oxidants and reductants are available at hundreds to thousands of meters depth. On the oxidizing side, CO2 and CO are available at considerable depths. This is of advantage to prokaryotes today that can generate acetate and an energy supply from acetate and also to other organisms that can use acetate. This is not too different from the chemiosmotic energygenerating system of The First Cell discussed above and not different at all when H2S is available. Life could have arisen and thrived shallow or deep underground (Gold, 1992), with sufficient depth to escape destructive cosmic radiation. So one might modify the dictum, from a ‘‘warm pond’’ to a ‘‘warm mud pie’’.

12. CONCLUSIONS How does one assemble all these processes, components, and thoughts coherently? We do so in a barely convincing manner today, but have made an

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attempt and hope that our effort serves useful purposes, especially in introducing this topic to a broad audience of microbiologists. As in all scientific questions, one should address the origin of The First Cell basically as a question of biological science. A clear exposition of the problems involved will help lead toward experimental tests of properties of single processes and components. Second, there is the heuristic value of providing a basis for classroom inclusion of this vital topic. The origin of cellular life is rarely considered as a topic in general microbiology texts, although it might be expected to play a central organizing role there.

ACKNOWLEDGEMENTS Microbial and molecular genetics developed in the authors’ time of conducting experimental research. A list of colleagues that exposed their significant ideas to us quickly grows too long; some colleagues are represented in the references below. The ideas from many sources, now compounded over the years, fit together quite well. Of course, drawing conclusions about the beginning of life is risky. The above description of the basics of what must have happened is largely due to the efforts of others, and only funneled through us. We thank our teachers and are grateful to them. We thank Le T. Phung for the figures and reference support and Leif Pallesen for redaction during revision.

REFERENCES Abramson, J., Iwata, S. and Kaback, H.R. (2004) Lactose permease as a paradigm for membrane transport proteins. Mol. Membr. Biol. 21, 227–236. Bernstein, M.P., Sandford, S.A. and Allamandola, L.J. (1999) Life’s far-flung raw materials. Sci. Amer. 281(1), 42–49. Boucher, Y. and Doolittle, W.F. (2000) The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways. Mol. Microbiol. 37, 703–716. Brack, A. (ed.). (1998) The Molecular Origins of Life: Assembling Pieces of the Puzzle. Cambridge University Press, Cambridge, UK. Cairns-Smith, A.G. (1990) Seven Clues to the Origin of Life: A Scientific Detective Story. Cambridge University Press, Cambridge, UK. Cavalier-Smith, T. (2001) Obcells as proto-organisms: membrane heredity, lithophosphorylation, and the origins of the genetic code, the first cells, and photosynthesis. J. Mol. Evol. 53, 555–595.

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