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Prokaryotes and lower eukaryotes
Prokaryotes and lower eukaryotes Room at the bottom: molecular insights from microbial organisms Editorial overview Ursula Goodenough and Terry Platt Washington University, St Louis, Missouri and University of Rochester Medical Center, Rochester, New York, USA Current Opinion in Genetics and Development 1992, 2:679--682 Introduction The impact of the molecular-biology revolution on our appreciation of the inter-relatedness of life cannot be overstated. With the glamour o f large-scale genome mapping and the successful creation of transgenic organisms, it is easy to lose sight of the continuing advances derived from studies of prokaryotes and lower eukaryotes, and their relevance to higher forms of life. Yanofsky [1], among others, has lucidly challenged such an oversight, echoing Feynman's earlier exhortation that we focus on 'the room at the bottom'. This issue serves to summarize some of the recent and exciting developments from these simple, but by no means simplistic, systems. Innumerable examples now exist of cloned mammalian genes that have homologs in yeast and/or Escherichia coli, with the known function of the 'lower' gene providing insight into the function of the 'higher', or vice versa. Similarly, as in vitro studies on reconstituted cellular activities become more sophisticated, the choice of system is largely dictated by which is the best characterized and most readily manipulated rather than by where it lies on the continuum from microbe to mammal. Synergism between powerful genetic tools and accessible biochemical approaches has also facilitated the understanding of many aspects of microbial cell function in unparalleled detail. The work reviewed here spans diverse areas: although it may appear 'a mile wide and an inch deep', we and our colleagues have tried to ensure that the freshness and importance of the reported developments counter-balance the necessary brevity and the exclusion of previous, established (albeit no less important) contributions from many laboratories. With only 18 reviews, comprehensive coverage of the subject is impossible and as such there are certain omissions (e.g. prokaryotic RNA splicing and transcriptional regulation) that we leave to be covered in future issues. In the tradition of the Current Opinion series, each review focuses on a particular topic. Where possible, we have encouraged the authors to consider both prokary-
otic and lower eukaryotic systems. In some cases, these contributions occur in pairs, whilst in others (e.g. nuclear trafficking), a direct comparison is futile. In this overview, we highlight some of the evolutionary comparisons that emerge from this collection of reviews. The topics chosen can be gathered into three categories. Approximately half of the reviews deal with genomes, their transcripts, and the mechanisms that have evolved to enhance either their structural reorganization or their control over expressing information. The second group encompasses transport and communication between cellular compartments and the interplay with cellular metabolism. The third category addresses issues of global control related to cellular division and the response to environmental challenges.
G e n o m e and transcript fluidity The first quartet of reviews, encompassing recombination and transposition, is an illuminating set, even as it sounds a theme for repetition and variation in the later articles. The past three decades of genetic studies in E. coli have revealed more than 20 genes involved in genetic recombination; most of these have now been cloned, sequenced and the specific proteins they encode have been identified (Lloyd and Sharpies, pp 683-690). Major recent advances have derived from systems that reconstitute the genetically predicted steps of recombination using purified proteins in vitro, confirming, for example, tile role of RecA protein in mediating heteroduplex joint formation and strand exchange, and showing that the RuvABC products catalyze branch migration and resolve Holliday junctions by endonucleolytic cleavage. The companion participation of many recombination genes in the repair of DNA damage, tile redundant roles of some genes, and the as yet unidentified function of others have, however, weakened the historical arguments for independent recombinational pathways. Developments in yeast follow
Abbreviations
ER-~endoplasmicreticulum; Fis--factor for inversion stimulation;hsp~heat-shockprotein; IHF--integration host factor; PAN--poly(A) nuclease;snRNP--small nuclear ribonucleoprotein;SRP--signalrecognitionparticle; tRNA--transferRNA. (~) Current Biology Ltd ISSN 0959-437X
679
680
Prokaryotes and lower eukaryotes closely, aided immensely by 'reverse genetics' used to identify and clone specific genes, and in particular to create new mutants (Strathem, pp 691-697). Correlations between the yeast genetic and physical map are proceeding apace, with distortions being used to define 'hot' (favoring recombination) and 'cold' spots in the genome. Proof has now been provided in vitro that synapsis is required for meiotic recombination, and that previous, genetically derived assumptions regarding mitotic crossing-over are biochemically valid. Of conceptual novelty is the emerging role of RNA-mediated recombination to account for intron loss, gene conversion and pseudogene formation. In a similar vein, the components of non-replicative and replicative transposition are also being elucidated (Hanfiord and Chaconas, pp 698-704). Stable macromolecular nucleoprotein complexes (including element-specific transposases) can now be isolated for study and in some cases may enable researchers to discriminate between the factors mediating synapsis and those participating in cleavage. The chemistry of nicking or double-strand cutting and strand traslsfer is now known, with the singlestep transesterification reaction possibly 'the paradigm for all transposition systems'. Transposition of the yeast retroviral Ty elements requires host proteins in addition to Ty-encoded factors (Sandmeyer, pp 705-711), but the biochemical evidence for participation of these proteins is still in its infancy, and the ability to mimic transposition in vitro is limited. The behavior of mutant proteins and target sites in vivo has been examined for both replication and transposition: a particularly interesting result is that disruption of the host DBR1 debranching enzyme depresses Tyl transposition by an unknown mechanism. Another major focus in Ty research is the limited copy number of these elements in the chromosome and the relationship of certain DNA-binding proteins to favored integration sites. The central role of RNA splicing in removing intervening intronic sequences from the transcripts of most eukaryotic and some prokaryotic genes is well known. The review by Woolford and Peebles (pp 712-719) focuses on spliceosome assembly and function, highlighting parallels with the protein-facilitated splicing of Group II and the recently defined Group III introns in Euglena chloroplasts. As an intensely studied ribonucleoprotein complex, the spliceosome ranks second only to the ribosome in complexity (and in historical seniority). The dynamic interplay among the components of the splicing reaction is a remarkable, choreographed ballet involving the U small nuclear ribonucleoproteins (snRNPs), host proteins and the newly appreciated aspects of RNA secondary and tertiary structure that affect both target alignment and the chemistry of splicing. It is gratifying that even as the variety of splicing examples increases, detailed in vitro studies are providing insights into generally applicable mechanisms, while continuing to raise challenges for the future. Independent of whether the primary transcript is modified by splicing, its translatability and lifetime have a critical affect on the expression of the mRNA-encoded
information. Unexpected intricacies of translational control are emerging in both prokaryotic and eukaryotic systems (Lindahl and Hinnebusch, pp 720-726). These include the modulation of secondary and tertiary mRNA structure (including hairpin loops and pseudoknots) by proteins functioning as translational repressors or activators. In prokaryotes, one example is the formation of a complex of mRNA and 30S particle that cannot initiate polypeptide biosynthesis. In yeast, evidence is mounting for two nuclear-encoded proteins, PET54 and CBS2, having a dual function in mitochondria: each promotes the translation of one specific mitochondrial gene and the splicing of a different transcript. Independent of mitochondrial expression, control of the global regulator GCN4 apparently depends on the phosphorylation of the initiation factor eIF-2 through a cascade stimulated initially by uncharged transfer RNA (tRNA). Advances in the complex and increasingly important area of mRNA turnover are reported by Higgins and Jacobson (pp 739-747). As is the case in recombination, E. coli has provided a wealth of information in vivo and in vitro in the form of enzymes that participate in mRNA degradation. The list has been extended to add the endonucleases designated RNaseE, -K, -M and -R to the old warhorse 3'-5' exonucleases, RNaselI and polynucleotide phosphorylase (no 5'-3' exonucleases are known in E. coli). The identification of alternative degradative pathways for different messenger molecules has come as less of a surprise than the discovery that turnover of a transcript can occur by an alternative pathway at a rate comparable to that of the primary pathway should it be blocked. Although general molecular mechanisms are probably conserved among prokaryotes and eukaryotes, the latter display some interesting conceptual differences: stability seems to be the default mode (in contrast to general mRNA instability in bacteria), and translation plays a more direct role in governing decay. In yeast, a number of tram-acting factors (e.g. UPF1 and -3, RNA14 and -15) have been implicated in affecting turnover, instability elements in several coding regions have been identified, decreased elongation rates have been shown to slow decay, while premature termination enhances it, and the poly(A) tail with its associated proteins, including the poly(A) nuclease (PAN), has an integral role in determining mRNA lifetime (in addition to its role in affecting translation initiation). The role of the 5'-3' exonuclease XRN1 is tantalizing but remains unresolved: XRN1 is also implicated in recombinational strand exchange and nuclear processing. Bacteriophage ~. is often considered as an excellent model of every facet of prokaryotic molecular biology. The isolation and characterization of ~. mutants has revealed much about the mechanisms of its lytic and lysogenic life cycles. It is less widely appreciated, but strongly re-emphasized in the review by Friedman (pp 727-738), that much can be leamed about the bacterial cell by examining mutations in tram-acting host functions that affect £ expression--often such mutations have obvious effects on ~ growth or expression although they do not affect the host. The roles of integration host factor (IHF) and factor for inversion stimulation (Fis) in binding and
Editorial overview Goodenough, Platt 681 bending DNA at their target sites during integrative or excisive recombination are now amenable to analysis in vitro. IHF is also implicated in transcriptional control, and the host Nus factors (including NusA, -B, -E and the newly characterized NusG factor) participate as RNAbinding and/or polymerase-binding proteins to modulate transcription elongation, in some cases by modifying the susceptibility of the polymerase core enzyme to other effector molecules. Of special interest (as replication is not covered elsewhere in this volume) is the discussion of the proteins, including DnaK, DnaJ, GrpE and GroE, that are involved in ~. replication and DNA packaging, and which fall into the 'heat-shock' protein (hsp) superfamily (see also Lindquist, pp 748-755) - - these are evidently chaperone proteins that can facilitate the refolding of denatured polypeptides.
Organelles and macromolecular complexes Flagellum Ironically, the major organdie with a con~'non name, the flagellum, shares no homology between prokaryotes and lower eukaryotes. Bacterial flagella are driven by a proton gradient and are rotated at the base to generate motility, while eukaryotic flagella are driven by ATP hydrolysis and undergo a bending motion (Blair and Dutcher, pp 756-767). Moreover, there is apparently no overlap in their polypeptide components. Rapid progress is being made in identifying the genes specifying particular flagellar components; Blair predicts that for bacteria the catalogue will be complete in a few years, whereas for Cblamydomonas, the eukaryotic model system for flagellar gene-protein correlation studies, it is a more distant goal. Despite the advanced level of molecular understanding, it remains unclear how the bacterial flagellum rotates and how the eukaryotic flagellum bends. Many models have, however, been forwarded and these stand or fall as confirmation is provided by the rapidly increasing genetic data. For example, the inner dynein arms of the eukaryotic flagellum, once absent from mechanistic models, are emerging as the key regulators of bend initiation and propagation, with the more conspicuous outer arms simply amplifying the power of the stroke.
Nucleus There is increasing appreciation that the nucleus has a life of its own and is in no sense a simple bag of chromosomes (Bossie and Silver, pp 768-774). The yeast nucleus emerges as a dynamic organelle, the protein import and RNA export of which are under elaborate regulation. Just as the Chlamydomonas flagellum is indistinguishable from a mammalian flagellum, so is the yeast nucleus highly homologous to a mammalian nucleus in its trafficking mechanisms and the gene-encoded products executing them. These products include nuclear import receptor proteins that bind targeted proteins in the cytoplasm and deliver them to the nucleus, perhaps in response to phosphorylation.
Endoplasmic reticulum Protein trafficking into the endoplasmic reticulum (ER) is thought to be homologous to protein export from E. coli, and a ribonucleoprotein complex termed the signal recognition particle (SRP) has long been considered essential to both processes (Fung et al., pp 775-779). Recently, however, a viable yeast strain has been engineered in which all SRP activity is deleted: it is moderately compromised, with growth rates at 25% of wild type. The controversy surrounding this finding underscores how a classic proposition can attain the status of central dogma: what's assumed to be true of E. coli and elephants must be true for yeast.
Channels
The secrets of channels are rapidly been uncovered. Preston et al. (pp 780-784) summarize recent intriguing research on ion channels. It is now documented that E. coli possesses mechanosensitive channels that are very similar to their fungal counterparts, and that bacterial cation channels (porins) share certain intriguing parallels with channels in the outer mitochondrial membrane. Potassium channels homologous to the shake~:encoded product of Drosophila have been detected in Paramecium and Arabidopsis (but not as yet in bacteria or yeast). In addition to ion channels, protein channels are coming to center stage. The SRP-mediated transit of proteins across the E. coli membrane has an apparent homolog in the eukaryotic 'translocon' that allows signal-carrying proteins to cross into the ER (Fung et a,(.). The n u c l e a r p o r e represents an analogous channel, gated in this case by nuclear-localization signals (Bossie and Silver).The nucleoporins lining the nuclear pores carry [B-sheet motifs that are reminiscent of the 13-sheet strands lining ion channels. Last but certainly not least is the channel that mediates bacterial flagellar assembly: a flagellum-specific pathway down the center of the growing organelle exports all the external components of the flagellum to their final location (Blair and Dutcher).
Global strategies The remaining articles in this volume consider more 'global' events such as cell-cycle control and spomlation. Discussion of prokaryote/eukaryote evolution must start with the central fact of endosymbiosis: mitochondria and chloroplasts clearly originated as resident bacteria and cyanobacteria, and Sogin [2] has recently speculated that the eukaryotic nucleus might itself have originated as an endosymbiotic archebacterium; that is, the genome itself might have been engulfed. The prokawotic lineage of chloroplasts is underscored in Rochaix's review (pp 785-791) of photosynthesis: current investigations move back and forth from free-living cyanobacteria to the chloroplasts of eukaryotic green algae, with the differences observed (in photosystem II stability, for example) minor as compared with the homologies.
682
Prokaryotes and lower eukaryotes If eukaryotes started out as integrated collections of prokaryotes, they have since developed their own approaches to existence. A particularly appealing generalization is the characterization of prokaryotes as 'chemical machines' and eukaryotes as 'morphogenetic machines' [3]. While the morphogenetic end-product (a fruiting body) of DicO~stelium is unimpressive in comparison with an eagle or oak tree, there is no question that most of the prerequisites for cell differentiation (includhag cytoskeletons and asymmetric cell divisions) were 'invented' by the lower eukaryotes; multicellularity and embryogenesis simply play out these potentialities in space and ~-ne. Consideration of the cell cycle and its control highlights fundamentally different strategies at work in prokaryotes and lower eukaryotes (Donachie, pp 792-798). E. coli strives to produce the maximum number of viable units per biomass in the shortest possible time, even if the results are a bit sloppy (e.g. the occasional generation of cells lacking DNA). Yeasts, in contrast, have evolved elaborate G2 controls to regulate cell size and have instituted careful checkpoints to ensure that DNA replication is complete before mitosis begins. The description of bacteria as chemical machines is apparendy contradicted by the elaborate sporulation programs of Bacillus subtilis and the myxobacteria that result in the generation of novel cell types. In fact, bacteria (and most simple eukaryotes) sporulate in response to starvation, when the chemical-machine option shuts down. Sporulation pathways in both prokaryotes and lower eukaryotes share an early requirement for prorein phosphorylation (Strauch and Hoch, pp 799-804), followed by major changes in gene expression. Novel cy factors are involved in B. subtilis, but in no system is there yet a clear picture of the signal-transduction pathways that elicit the switches in transcription; to borrow the authors' pun, this represents a fruitful area of research. Just as sporulation in simple organisms under nutritional stress 'makes sense', so does the ubiquitous heat-shock response (Lindquist) and, in fact, starvation and other stresses prove to induce the heat-shock pathways as well. The eukaryotic hsps that cooperate to mediate the refolding or the disposal of denatured proteins are in some cases directly homologous to their prokaryotic forebears, while in other cases novel pathways have evolved. As noted earlier, hsps may also participate in additional cellular activities, such as X DNA replication and general chaperone functions. Pathogenic and parasitic infection represents a particulady crucial interface between microbes and multicel-
lular organisms, and two reviews consider this topic. Lindquist cites the role of hsps in buffering the transition of a pathogen to its 37oc host, and notes that several (auto)immune responses are directed at hsps. Swanson, Belland and Hill (pp 805-811) consider the "surface variation" displayed by many parasitic organisms. They note that similar recombinational switching strategies are employed to generate new surface proteins whether the pathogen is prokaryotic or eukaryotic, although the rationales differ. In bacteria (e.g. Neisseria gonorrhoeae), surface variation is used to optimize infection, whereas in eukaryotic parasites (e.g. trypanosomes, borelliae) the process is used to foil the immune system and thus prolong a chronic infection.
Conclusions In the hopes of bridging the taxonomically imposed but artificial chasm between prokaryotes and eukaryotes, this collection of reviews is intended to compare and contrast the biological solutions to cellular problems among single-cell organisms on the two sides of this gulf. In addition, most of the articles exemplify how the interplay between genetics and biochemistry is aiding the expansion of our knowledge. It comes as no surprise that in general the conceptual similarities far outweigh the organismic differences. Whether the inference that higher eukaryotes are likely to share many of the same characteristics is valid remains to be determined, but if this volume provides some useful ideas and testable hypotheses for the study of metazoan systems, it will have provided an unanticipated bonus quite apart from its obvious target.
References YANOFSKYC: What Will be the Fate of Research otes? Cell 1991, 65:199-200.
on
Prokary-
SOGIN MIz Early Evolution and the Origin of Curr Opin Genet Dev 1991, 1:457-463.
Eukaryotes.
LAMPORTDTA: Structure and Function of Glycoproteins. In The Biochemistry of Plants, Vol 3. Edited by Preiss J. New York: Academic Press; 1980:501-541. U Goodenough, Department of Biology, Washington University, 1 BrookingsDrive,St Louis,Missouri63130-4899, USA. T Platt, Department of Biochemistry,Universityof Rochester Medical Center, 601 ElmwoodAvenue, Rochester,New York 14642, USA.
otic and lower eukaryotic systems. In some cases, these contributions occur in pairs, whilst in others (e.g. nuclear trafficking), a direct comparison is futile. In this overview, we highlight some of the evolutionary comparisons that emerge from this collection of reviews. The topics chosen can be gathered into three categories. Approximately half of the reviews deal with genomes, their transcripts, and the mechanisms that have evolved to enhance either their structural reorganization or their control over expressing information. The second group encompasses transport and communication between cellular compartments and the interplay with cellular metabolism. The third category addresses issues of global control related to cellular division and the response to environmental challenges.
G e n o m e and transcript fluidity The first quartet of reviews, encompassing recombination and transposition, is an illuminating set, even as it sounds a theme for repetition and variation in the later articles. The past three decades of genetic studies in E. coli have revealed more than 20 genes involved in genetic recombination; most of these have now been cloned, sequenced and the specific proteins they encode have been identified (Lloyd and Sharpies, pp 683-690). Major recent advances have derived from systems that reconstitute the genetically predicted steps of recombination using purified proteins in vitro, confirming, for example, tile role of RecA protein in mediating heteroduplex joint formation and strand exchange, and showing that the RuvABC products catalyze branch migration and resolve Holliday junctions by endonucleolytic cleavage. The companion participation of many recombination genes in the repair of DNA damage, tile redundant roles of some genes, and the as yet unidentified function of others have, however, weakened the historical arguments for independent recombinational pathways. Developments in yeast follow
Abbreviations
ER-~endoplasmicreticulum; Fis--factor for inversion stimulation;hsp~heat-shockprotein; IHF--integration host factor; PAN--poly(A) nuclease;snRNP--small nuclear ribonucleoprotein;SRP--signalrecognitionparticle; tRNA--transferRNA. (~) Current Biology Ltd ISSN 0959-437X
679
680
Prokaryotes and lower eukaryotes closely, aided immensely by 'reverse genetics' used to identify and clone specific genes, and in particular to create new mutants (Strathem, pp 691-697). Correlations between the yeast genetic and physical map are proceeding apace, with distortions being used to define 'hot' (favoring recombination) and 'cold' spots in the genome. Proof has now been provided in vitro that synapsis is required for meiotic recombination, and that previous, genetically derived assumptions regarding mitotic crossing-over are biochemically valid. Of conceptual novelty is the emerging role of RNA-mediated recombination to account for intron loss, gene conversion and pseudogene formation. In a similar vein, the components of non-replicative and replicative transposition are also being elucidated (Hanfiord and Chaconas, pp 698-704). Stable macromolecular nucleoprotein complexes (including element-specific transposases) can now be isolated for study and in some cases may enable researchers to discriminate between the factors mediating synapsis and those participating in cleavage. The chemistry of nicking or double-strand cutting and strand traslsfer is now known, with the singlestep transesterification reaction possibly 'the paradigm for all transposition systems'. Transposition of the yeast retroviral Ty elements requires host proteins in addition to Ty-encoded factors (Sandmeyer, pp 705-711), but the biochemical evidence for participation of these proteins is still in its infancy, and the ability to mimic transposition in vitro is limited. The behavior of mutant proteins and target sites in vivo has been examined for both replication and transposition: a particularly interesting result is that disruption of the host DBR1 debranching enzyme depresses Tyl transposition by an unknown mechanism. Another major focus in Ty research is the limited copy number of these elements in the chromosome and the relationship of certain DNA-binding proteins to favored integration sites. The central role of RNA splicing in removing intervening intronic sequences from the transcripts of most eukaryotic and some prokaryotic genes is well known. The review by Woolford and Peebles (pp 712-719) focuses on spliceosome assembly and function, highlighting parallels with the protein-facilitated splicing of Group II and the recently defined Group III introns in Euglena chloroplasts. As an intensely studied ribonucleoprotein complex, the spliceosome ranks second only to the ribosome in complexity (and in historical seniority). The dynamic interplay among the components of the splicing reaction is a remarkable, choreographed ballet involving the U small nuclear ribonucleoproteins (snRNPs), host proteins and the newly appreciated aspects of RNA secondary and tertiary structure that affect both target alignment and the chemistry of splicing. It is gratifying that even as the variety of splicing examples increases, detailed in vitro studies are providing insights into generally applicable mechanisms, while continuing to raise challenges for the future. Independent of whether the primary transcript is modified by splicing, its translatability and lifetime have a critical affect on the expression of the mRNA-encoded
information. Unexpected intricacies of translational control are emerging in both prokaryotic and eukaryotic systems (Lindahl and Hinnebusch, pp 720-726). These include the modulation of secondary and tertiary mRNA structure (including hairpin loops and pseudoknots) by proteins functioning as translational repressors or activators. In prokaryotes, one example is the formation of a complex of mRNA and 30S particle that cannot initiate polypeptide biosynthesis. In yeast, evidence is mounting for two nuclear-encoded proteins, PET54 and CBS2, having a dual function in mitochondria: each promotes the translation of one specific mitochondrial gene and the splicing of a different transcript. Independent of mitochondrial expression, control of the global regulator GCN4 apparently depends on the phosphorylation of the initiation factor eIF-2 through a cascade stimulated initially by uncharged transfer RNA (tRNA). Advances in the complex and increasingly important area of mRNA turnover are reported by Higgins and Jacobson (pp 739-747). As is the case in recombination, E. coli has provided a wealth of information in vivo and in vitro in the form of enzymes that participate in mRNA degradation. The list has been extended to add the endonucleases designated RNaseE, -K, -M and -R to the old warhorse 3'-5' exonucleases, RNaselI and polynucleotide phosphorylase (no 5'-3' exonucleases are known in E. coli). The identification of alternative degradative pathways for different messenger molecules has come as less of a surprise than the discovery that turnover of a transcript can occur by an alternative pathway at a rate comparable to that of the primary pathway should it be blocked. Although general molecular mechanisms are probably conserved among prokaryotes and eukaryotes, the latter display some interesting conceptual differences: stability seems to be the default mode (in contrast to general mRNA instability in bacteria), and translation plays a more direct role in governing decay. In yeast, a number of tram-acting factors (e.g. UPF1 and -3, RNA14 and -15) have been implicated in affecting turnover, instability elements in several coding regions have been identified, decreased elongation rates have been shown to slow decay, while premature termination enhances it, and the poly(A) tail with its associated proteins, including the poly(A) nuclease (PAN), has an integral role in determining mRNA lifetime (in addition to its role in affecting translation initiation). The role of the 5'-3' exonuclease XRN1 is tantalizing but remains unresolved: XRN1 is also implicated in recombinational strand exchange and nuclear processing. Bacteriophage ~. is often considered as an excellent model of every facet of prokaryotic molecular biology. The isolation and characterization of ~. mutants has revealed much about the mechanisms of its lytic and lysogenic life cycles. It is less widely appreciated, but strongly re-emphasized in the review by Friedman (pp 727-738), that much can be leamed about the bacterial cell by examining mutations in tram-acting host functions that affect £ expression--often such mutations have obvious effects on ~ growth or expression although they do not affect the host. The roles of integration host factor (IHF) and factor for inversion stimulation (Fis) in binding and
Editorial overview Goodenough, Platt 681 bending DNA at their target sites during integrative or excisive recombination are now amenable to analysis in vitro. IHF is also implicated in transcriptional control, and the host Nus factors (including NusA, -B, -E and the newly characterized NusG factor) participate as RNAbinding and/or polymerase-binding proteins to modulate transcription elongation, in some cases by modifying the susceptibility of the polymerase core enzyme to other effector molecules. Of special interest (as replication is not covered elsewhere in this volume) is the discussion of the proteins, including DnaK, DnaJ, GrpE and GroE, that are involved in ~. replication and DNA packaging, and which fall into the 'heat-shock' protein (hsp) superfamily (see also Lindquist, pp 748-755) - - these are evidently chaperone proteins that can facilitate the refolding of denatured polypeptides.
Organelles and macromolecular complexes Flagellum Ironically, the major organdie with a con~'non name, the flagellum, shares no homology between prokaryotes and lower eukaryotes. Bacterial flagella are driven by a proton gradient and are rotated at the base to generate motility, while eukaryotic flagella are driven by ATP hydrolysis and undergo a bending motion (Blair and Dutcher, pp 756-767). Moreover, there is apparently no overlap in their polypeptide components. Rapid progress is being made in identifying the genes specifying particular flagellar components; Blair predicts that for bacteria the catalogue will be complete in a few years, whereas for Cblamydomonas, the eukaryotic model system for flagellar gene-protein correlation studies, it is a more distant goal. Despite the advanced level of molecular understanding, it remains unclear how the bacterial flagellum rotates and how the eukaryotic flagellum bends. Many models have, however, been forwarded and these stand or fall as confirmation is provided by the rapidly increasing genetic data. For example, the inner dynein arms of the eukaryotic flagellum, once absent from mechanistic models, are emerging as the key regulators of bend initiation and propagation, with the more conspicuous outer arms simply amplifying the power of the stroke.
Nucleus There is increasing appreciation that the nucleus has a life of its own and is in no sense a simple bag of chromosomes (Bossie and Silver, pp 768-774). The yeast nucleus emerges as a dynamic organelle, the protein import and RNA export of which are under elaborate regulation. Just as the Chlamydomonas flagellum is indistinguishable from a mammalian flagellum, so is the yeast nucleus highly homologous to a mammalian nucleus in its trafficking mechanisms and the gene-encoded products executing them. These products include nuclear import receptor proteins that bind targeted proteins in the cytoplasm and deliver them to the nucleus, perhaps in response to phosphorylation.
Endoplasmic reticulum Protein trafficking into the endoplasmic reticulum (ER) is thought to be homologous to protein export from E. coli, and a ribonucleoprotein complex termed the signal recognition particle (SRP) has long been considered essential to both processes (Fung et al., pp 775-779). Recently, however, a viable yeast strain has been engineered in which all SRP activity is deleted: it is moderately compromised, with growth rates at 25% of wild type. The controversy surrounding this finding underscores how a classic proposition can attain the status of central dogma: what's assumed to be true of E. coli and elephants must be true for yeast.
Channels
The secrets of channels are rapidly been uncovered. Preston et al. (pp 780-784) summarize recent intriguing research on ion channels. It is now documented that E. coli possesses mechanosensitive channels that are very similar to their fungal counterparts, and that bacterial cation channels (porins) share certain intriguing parallels with channels in the outer mitochondrial membrane. Potassium channels homologous to the shake~:encoded product of Drosophila have been detected in Paramecium and Arabidopsis (but not as yet in bacteria or yeast). In addition to ion channels, protein channels are coming to center stage. The SRP-mediated transit of proteins across the E. coli membrane has an apparent homolog in the eukaryotic 'translocon' that allows signal-carrying proteins to cross into the ER (Fung et a,(.). The n u c l e a r p o r e represents an analogous channel, gated in this case by nuclear-localization signals (Bossie and Silver).The nucleoporins lining the nuclear pores carry [B-sheet motifs that are reminiscent of the 13-sheet strands lining ion channels. Last but certainly not least is the channel that mediates bacterial flagellar assembly: a flagellum-specific pathway down the center of the growing organelle exports all the external components of the flagellum to their final location (Blair and Dutcher).
Global strategies The remaining articles in this volume consider more 'global' events such as cell-cycle control and spomlation. Discussion of prokaryote/eukaryote evolution must start with the central fact of endosymbiosis: mitochondria and chloroplasts clearly originated as resident bacteria and cyanobacteria, and Sogin [2] has recently speculated that the eukaryotic nucleus might itself have originated as an endosymbiotic archebacterium; that is, the genome itself might have been engulfed. The prokawotic lineage of chloroplasts is underscored in Rochaix's review (pp 785-791) of photosynthesis: current investigations move back and forth from free-living cyanobacteria to the chloroplasts of eukaryotic green algae, with the differences observed (in photosystem II stability, for example) minor as compared with the homologies.
682
Prokaryotes and lower eukaryotes If eukaryotes started out as integrated collections of prokaryotes, they have since developed their own approaches to existence. A particularly appealing generalization is the characterization of prokaryotes as 'chemical machines' and eukaryotes as 'morphogenetic machines' [3]. While the morphogenetic end-product (a fruiting body) of DicO~stelium is unimpressive in comparison with an eagle or oak tree, there is no question that most of the prerequisites for cell differentiation (includhag cytoskeletons and asymmetric cell divisions) were 'invented' by the lower eukaryotes; multicellularity and embryogenesis simply play out these potentialities in space and ~-ne. Consideration of the cell cycle and its control highlights fundamentally different strategies at work in prokaryotes and lower eukaryotes (Donachie, pp 792-798). E. coli strives to produce the maximum number of viable units per biomass in the shortest possible time, even if the results are a bit sloppy (e.g. the occasional generation of cells lacking DNA). Yeasts, in contrast, have evolved elaborate G2 controls to regulate cell size and have instituted careful checkpoints to ensure that DNA replication is complete before mitosis begins. The description of bacteria as chemical machines is apparendy contradicted by the elaborate sporulation programs of Bacillus subtilis and the myxobacteria that result in the generation of novel cell types. In fact, bacteria (and most simple eukaryotes) sporulate in response to starvation, when the chemical-machine option shuts down. Sporulation pathways in both prokaryotes and lower eukaryotes share an early requirement for prorein phosphorylation (Strauch and Hoch, pp 799-804), followed by major changes in gene expression. Novel cy factors are involved in B. subtilis, but in no system is there yet a clear picture of the signal-transduction pathways that elicit the switches in transcription; to borrow the authors' pun, this represents a fruitful area of research. Just as sporulation in simple organisms under nutritional stress 'makes sense', so does the ubiquitous heat-shock response (Lindquist) and, in fact, starvation and other stresses prove to induce the heat-shock pathways as well. The eukaryotic hsps that cooperate to mediate the refolding or the disposal of denatured proteins are in some cases directly homologous to their prokaryotic forebears, while in other cases novel pathways have evolved. As noted earlier, hsps may also participate in additional cellular activities, such as X DNA replication and general chaperone functions. Pathogenic and parasitic infection represents a particulady crucial interface between microbes and multicel-
lular organisms, and two reviews consider this topic. Lindquist cites the role of hsps in buffering the transition of a pathogen to its 37oc host, and notes that several (auto)immune responses are directed at hsps. Swanson, Belland and Hill (pp 805-811) consider the "surface variation" displayed by many parasitic organisms. They note that similar recombinational switching strategies are employed to generate new surface proteins whether the pathogen is prokaryotic or eukaryotic, although the rationales differ. In bacteria (e.g. Neisseria gonorrhoeae), surface variation is used to optimize infection, whereas in eukaryotic parasites (e.g. trypanosomes, borelliae) the process is used to foil the immune system and thus prolong a chronic infection.
Conclusions In the hopes of bridging the taxonomically imposed but artificial chasm between prokaryotes and eukaryotes, this collection of reviews is intended to compare and contrast the biological solutions to cellular problems among single-cell organisms on the two sides of this gulf. In addition, most of the articles exemplify how the interplay between genetics and biochemistry is aiding the expansion of our knowledge. It comes as no surprise that in general the conceptual similarities far outweigh the organismic differences. Whether the inference that higher eukaryotes are likely to share many of the same characteristics is valid remains to be determined, but if this volume provides some useful ideas and testable hypotheses for the study of metazoan systems, it will have provided an unanticipated bonus quite apart from its obvious target.
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