Polymerase evolution and organism evolution Brian Palenik University of California, San Diego, La Jolla, USA The continuing exploration of the structure-function relationships of polymerases and the use of polymerases as phylogenetic tools complement each other, as seen in the literature for the past year. DNA-dependent RNA-polymerase gene sequences, in particular, have been used both to define functional domains in the protein encoded and recently to explore fundamental questions in evolution. Current Opinion in Genetics and Development 1992, 2:931-936

Introduction Studies of protein structure-function relationships and the evolution of organisms are subtly intertwined. It is common practice to use sequence data from diverse organisms to define highly conserved domains in proteins and to develop models of protein 'motifs'; in effect the evolution of organisms is exploited to further our understanding of proteins. The other side of the coin is that protein sequence data are now also frequently used to elucidate the evolution of organisms from the population to the kingdom level. In these studies, it is vital to know the location of highly conserved domains. Sequences from these domains are themselves used to determine the relationship between highly diverged organisms as they are the only sequence data that can be unambiguously aligned. These regions can also be used to develop primers that are used in PCR to obtain sequence data from related groups of organisms. There has been and will continue to be a fertile interchange between molecular evolution and biological chemistry. The various polymerases (DNA, RNA and reverse transcriptase) are the subject of numerous studies from many different perspectives. While studies of their biochemical characteristics are progressing rapidly, their DNA or derived amino-acid sequences are also increasingly being used as phylogenetic tools. The purpose of this paper is to review the recent literature in both of these areas in the hope of illuminating their common ground. First, I will review the current picture of the structure-function relationship of DNA-dependent RNA polymerase (earlier reviews should also be consulted [1-3]). Second, I will assess the recent use of this enzyme in studying the evolution of various organisms, and finally I shall highlight some of the other polymerases that are being used as phylogenetic tools. Unfortunately, the corresponding functional studies of other polymerases are beyond the scope of this review.

The largest subunit of DNA-dependent RNA polymerase: ~3' The largest subunit of Escherichia coli DNA-dependent RNA polymerase, 13', and its equivalent subunit in other prokaryotes and the eukaryotes, is thought to be involved in template binding. Beginning with the first available DNA sequences of this subunit, efforts have been made to define highly conserved regions and their functions. One of the earliest papers [4] defined six regions, but in the most comprehensive alignment to date, Iwabe et al. [5"] have defined nine such regions (Fig. 1 ) - - regions 2, 3, 4, 6, 7 and 9 correspond to the previous regions 1-6, respectively. Recendy, further sequences for the largest subunit have become available, including that of Giardia lamblia RNA polymerase (pol) I11 [6], and human [7], Arabidopsis [8] and Caenorhabditis [9] pol I1, but these sequences do not alter the existing definitions of the most highly conserved regions. Some of the recent work to define the potential functions of these regions has concentrated on potential zinc-binding domains, as the [3' subunit has been shown to bind zinc (reviewed in [10]). Using a protein-expression system and a zinc-blotting technique, the amino-terminal region of the largest subunit in yeast pol II (residues 47-119) was shown to bind zinc in vitro [11..]. One problem is that this region contains in total nine cysteines and histidines that appear to be relatively conserved, and the particular residues binding zinc have still not been defined. The 'redundancy' of these amino acids could potentially be important. If a mutation occurs in the codon for one cysteine, perhaps a neighboring cysteine can be substituted; or alternatively, perhaps the conformation of polymerase is affected by which of the particular residues bind zinc. In E. coli and other prokaryotes, the 13' subunit also binds zinc. Again, the precise amino acids have not been defined, but a highly conserved zinc-finger motif,

Abbreviation pol--polymerase.

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ally involved in RNA chain translocation. Mutations found in the DNA-dependent RNA-polymerase [3 subunit that suppress tile phenotype of mutations in 13' suggest that both the amino- and carboxy-terminal regions of 13' may interact with the carboxy-terminal region (including the zinc-binding domain, see below) of [3 [17,18..]. The eukaryotic RNA pol II largest subunits have multiple copies of a heptapeptide repeat, Tyr-Ser-Pro-ThrSer-Pro-Ser, at tile carboxy-terminal end [4], which is phosphorylated to varying degrees. The function of this domain is apparently to ensure accurate transcription initiation from a subset of genes [19.,20"].

CysXCysX12_18CysX2Cys (where X represents any amino acid), is an obvious candidate [12",13.]. Surprisingly, in contrast to E. coli ~', which binds one zinc, the 13' equivalent of Bacillus subtilis is thought to bind tightly to two molecules of zinc (reviewed in [10]). Unfortunately, the sequence data from B. subtilis that might help in localizing the zinc-binding domains is not available. Potential DNA-binding helix motifs have also been noted in 13',consistent with its role in template binding (see Fig. 1). These motifs share some homology to helices J, K and P in DNA polymerases [4]. The functions of most other domains, some of which are extremely conserved (for example, Tyr-Asn-Ala-AspPhe-Asp-Gly-Asp-Glu/Gln-Met in region 4), are not understood. Recently, however, a number of mutants have been isolated or created in 13' whose phenotypes (ranging from inositol auxotrophy to increased chromosomal copy number) provide clues to the functions of the largest subunit's various domains [ 14".,15-]. Sensitivit3, to ~-amanitin, a fungal cyclic peptide that selectively inhibits pol II by blocking RNA chain elongation after phosphodiester bond formation, was reduced in a mouse cell-line by a mutation changing an asparagine residue found in pol II to an aspartic acid [16]. This residue occurs in the highly conserved domain 5, which might thus be gener-

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Fig. 1. The amino-acid sequence of the Escherichia coil RNA polymerase [3' subunit, rpoC (the scale represents amino acids). (a) Schematic representation of the conserved regions (numbered white boxes) and (b) possible functions of the largest subunit of DNA-dependent RNA polymerases in terms of E. coil ~J'.

The second largest subunit:

The second largest subunit of E. colt RNA poly/nerase, 13, and its equivalents in other organisms, appear to carry the catalytic site for phosphodiester-bond formation and to be involved in RNA chain initiation and elongation. The alignment of many of the available sequences for the 'second largest' subunits of RNA polymerases defines 9-12 regions of homology. The nine regions (A-I) as defined by Sweetser et al. [21] correspond to the regions 1,3,5,7-12 of Iwabe et al. [5"'] (see Fig. 2).

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Fig. 2. Schematic representation the amino-acid sequence of the Escherichia coil RNA polymerase ~ subunit, rpoB (the scale represents amino acids). (a) The conserved regions (numbered white boxes} and some recently characterized mutations (indicated below the sequence) are shown. (b) Possible functions of the second largest subunit of DNA-dependent RNA polymerases in terms of E. coil ~J.

Polymerase evolution and organism evolution Palenik 933. Mutations in the E. coli f~ subunit or its eukaryotic equivalents have recently been characterized in regions 6 [22.], 8 [23"], 9 [24], 11 [25"], and 12 [25"] (Fig. 2). Rifampicin-resistance mutants have been mapped to regions 6 and 7 [26]; toge.ther these are probably involved in nascent RNA chain stabilization or translocation. Region 10 has a nucleotide-binding motif [21]. Region 12 and sequences further downstream contain zinc-binding motifs in eukaryotes. Site-directed mutagenesis and zinoblotting techniques suggest that residues Cys1163, Cys1166, Cys1182 and Cys1185 (yeast pol II notation) are involved in zinc-binding [11..]. Results from studies of deletions between regions 10 and 11 in E. coli suggest that this region is not essential [27"]: here, the proteolytic cleavage of 13 into two pieces does not affect reconstitution of the enzyme [28-]. A recent paper suggests that just upstream of region 5 there is significant homology between bacterial RNases and eukaryotic RNA polymerases [29"]. This would suggest that some RNases may have been derived from RNA polymerases, or that eukaryotic but not eubacterial RNA polymerases have retained an RNase-like activity that has not yet been demonstrated experimentally.

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Fig. 3. Schematic representation of the c, subunit of Escherichia coil DNA-dependent RNA polymerase (the scale represents amino acids). (a) The conserved regions relative to chloroplast (white box), and some recently characterized mutations are shown. (b) Possible functions of the 0t subunit are indicated.

Transcription factors and c~ factors The smallest subunit: Although in the past, ct, the smallest subunit of E. coli RNA polymerase (Fig. 3), has received comparatively little attention, this situation is changing. Amino-acid alignments of available ~poA sequences suggest that approximately half of the protein, the amino-terminal end, is relatively conserved, in particular residues 24-123. The 0t subunit is probably involved in RNA polymerase assembly [3], and temperature-sensitive mutations that block assembly at non-permissive temperatures have been found in these conserved regions [30]. The anlino-terminal region may interact with a second et subunit or with conserved regions in the other subunits to promote assembly. The relationship of the ~x subunit, if any, to the many polypeptides purified from eukaryotic RNA polymerases has been puzzling. Recent results suggest that eukaryotic homologues of et do exist. Dequard-Chablat et al. [31"] have recently shown a striking homology between a and the AC19, AC40, and B44.5 subunits of yeast RNA polymerases. This homology appears to extend even further downstream of the region characterized in their work. The carboxy-terminal domain of 0t in E. coli, B. subtilis and green-plant chloroplasts is not well conserved, except for a small region around amino acids 262-271. Mutations in the carboxy-terminal domain that affect the transcriptional activation of a subset of positively regulated operons have been recently characterized [32"-35"]. These mutations suggest that there are interactions between this subunit and a subset of transcription factors and therefore this region is not expected to be strongly conserved between chloroplasts and E coll.

Transcription specificity is typically conferred on RNA polymerases by cr factors and transcription factors. The conserved domains in bacterial cr factors have been discussed elsewhere [36] and have recently been updated [37"]. The regions that are possibly conserved between bacterial c factors, the yeast mitochondrial transcription factor (derived from a virus?), and various eukaryotic transcription factors are currently the subjects of intensive investigation [38o-40.,41o-].

Phylogenetic studies using DNA-dependent RNA polymerases A number of recent papers have used DNA-dependent RNA-polymerase sequences to explore evolutionary quest_ions. One issue has been the evolution of the eukaryotic polymerases themselves. As eubacteria and archaebacteria have only one RNA polymerase, the mechanism(s) by which the three eukaryotic polymerases arose have been a matter of speculation. Early on, M~met et al. [42] noted that pol II, synthesizing mRNA, and pol III, synthesizing 5S and tRNAs, are more closely related to each other than either is to pol I, which synthesizes rRNAS. A paper by P0hler et al. [43] (also one of the first to use polymerase sequence data to derive phylogenetic trees) used sequences from pol I (Saccharomyces cerevisiae), pol II (S. cerevisiae, Trypanosoma brucei) and pol 1II (S. cereviaiae, T. brucei), the three major groups of archaebacteria, E. coli, and chloroplasts, to suggest that pol I is more closely related to eubacterial sequences and may have arisen from a eubacterial component of the

934

Genomesand evolution first eukaryote, lwabe et at [5"'], have more recently revisited this question and maintain that this is not the case. Convincingly, they suggest that pol I has diverged more rapidly than pol II or pol III, and thus it would be difficult to tree this gene correctly using maximum parsimony methods. Using a maximum-likelihood approach they found the eukaryotic polymerases grouped together, with bootstrap values around 74-85%. It would be useful to include a pol I sequence from T. b r u c e i o r other organisms in their analysis as this enzyme was only represented by yeast sequence data. Both Ptihler et al. [43] and Iwabe et al. [5"] address another fundamental issue in evolution: the origin of eukaryotes. The debate centers on whether tile archaebacteria are monophyletic or whether the eukaryotes specifically branch from one of the groups of archaebacteria, the 'eocytes'. Both papers suggest that, based on the sequences of the largest subunit of RNA polymerase, the archaebacteria group together. The archaebacteria, in addition, all have a split 'largest subunit gene' (see Fig. 1) that is not found in eukaryotes. This fact requires that, for the eocTte tree to be true, the eukaryotic gene would either have had to 'fuse back together' early on or tile split in the eocTtes must have fortuitously occurred at the same point as that in the other two archaebacterial groups. The latter possibility is obx4ously less likely than the former. Although both papers agree, gene sequence data from EF-lc~ has recently been used to argue that the archaebacteria are not monophyletic, as based on an eleven amino-acid insertion [44]. Interestingly, the analysis of data from the second largest polymerase subunit is less conclusive in elucidating the origin of eukaryotes, with either the eocTte tree or the archaebacterial tree being possible. Unfortunately, data from the second largest subunit of pol I and III, which might affect the results, are not yet available. However, sequence data are accumulating so rapidly that data now exists for other pol I largest subunits and the second largest polymerase subunits from a number of organisms [45,46]. Thus, the questions of the development of pol 1, II, and III, and the issue of the origin of the eukaryotes should be reassesed soon. In any event, both [5"] and [43] describe provocative applications of polymerase sequence data to clarify controversial evolutionary questions. Another evolutionary issue to which RNA polymerase sequence data has been applied is the origin of the chloroplast.. Sequence data alone, and the fact that both cyanobacteria and chloroplasts have a split largest subunit, yielding rpoC1 and rpoC2 (see Fig. 1), supports the endosymbiotic origin of the chloroplast from a cyanobacterium-like organism [12-]. One wrinkle in this issue was whether or not the endosy'mbiont was a prochlorophyte, already containing chlorophyll b, or a cyanobacterium, which uses phycobiliproteins as lightharvesting pigments. RNA polymerase sequence data was obtained from the three known prochlorophyte lineages by using PCR and conserved rpoC1 primers and has shown that none of the known lineages is specifically related to the lineage of the green chloroplast ancestor [13"]. In addition, it is demonstrated that prochloro-

phytes do not to share a common chlorophyll b containing (prochlorophyte) ancestor. The presence of highly conserved domains in RNA polymerases make the corresponding genes logical candidates for evolutionary studies that use PCR techniques to obtain sequence data. During the past year, DNA-dependent RNA polymerases have also been used to explore the evolution of linear DNA plasmids [47..]. RNA polymerase suburtits that are common only to eukaryotes and archaebacteria have been found [48], providing further insight into the evolution of these organisms. A gene tree for the various bacterial RNA polymerase c~ factors has been constructed [37"], with interesting implications for the roles of alternative cs factors in the evolution of bacterial developmental pathways. Clearly, the use of sequence data from RNA polT~llerase subunits to answer various evolutionary questions is rapidly expanding.

Evolutionary studies with other polymerases Other polymerases are also increasingly being used as efficient tools for understanding the evolution of organisms. Polymerases are some of the few highly conserved enzymes in diverse groups of viruses and are useful plwlogenetic tools. Polymerase (.L-protein) sequences have been used to group non-segmented negative-strand RNA viruses [49"]. RNA-dependent RNA-polymerase genes have recently been used to study and group positivestrand RNA viruses [50"] ,and evolutionary divergence of these viruses has been used to define the essential domains of tile polymerase [51"]. DNA-dependent DNA polymerase have ,also been used to clarify linear plasmid and viral evolution [47"'], and research using DNA polymerases and reverse transcriptases to study more ancient divergences is in process [52,53]. A general criticism is that too often there is insufficient justification using bootstrap or other statistical analyses for the phylogenetic tree chosen from the forest.

Conclusion Polymerase sequences have proven to be powerful phylogenetic tools. Surprisingly, they have not yet been exploited to assess population and species level distinctions. As more data become available, however, the presence in polymerase genes of highly conserved regions flanking less evolutionarily constrained regions (e.g. the E. coli ~J subunit), together with the use of PCR, will make polymerases useful in resolving evolutionary questions in these areas as well.

Acknowledgements The writing of this review was supported by ONR Grant N00014-91-J-1541 to J Swift and Department of Molecular Genetics

Polymerase evolution and organism evolution Palenik 935 and Cell Biology, University of Chicago. I also thank R Haselkom and B Brahamsha for comments on the manuscript.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of oustanding interest 1.

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2.

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ALLISON'LA, MOYI.E M, SHALES M, INGLES CJ: Extensive Homology Among the Largest Subunits of Eukaryotic and Prokaryotic RNA Polymerases. Cell 1985, 42:599~510.

5. ••

IWABE N, KUMA K, KISHINO H, I-b~SEGAWAM, MIYATAT: Evolution of RNA Polymerases and Branching Patterns of the Three Major Groups of Archaebacteria. J Mol Evol 1991, 32:70-78. Building on the work of M6met [421 and P/.ihler [43], the authors use DNA-dependent RNA-polymerase sequences to address the origins of the three eukaryotic polymerases and the origins of eukaryotes themselves. The paper details alignments of the conseta,ed amino acids for the two largest subunits. 6.

LANZF.NDORFERM, PAD,I P, GRAMI'I' B, PEATnE DA, ZILI.IG W: Nucleotide Sequence of the Gene Encoding t h e Largest Subunit of the DNA-dependent RNA Polymerase 111 of Gi. ardia lamblia. Nucleic Acids Res 1992, 20:1145.

7.

WINTZERITHM, ACKER J, VICAIRE S, VIGNERON M, KEDINGER C: Complete Sequence of the H u m a n RNA Polymerase II Largest Subunit. Nucleic Acids Res 1992, 20:910.

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DIETRICHMA, PRENGERJP, GUILFOYLETJ: Analysis of the G e n e s Encoding the Largest Subunit of RNA Polymerase II in Ara. bidopsis and Soybean. Plant Mol Biol 1990, 15:207-223.

9.

BIRD DM, PdDDI.E DL: Molecular Cloning and Sequencing of a m a . l , the Gene Encoding t h e Largest Subunit of Caenorbabditis elegans RNA polymerase II. Mol Cell Biol 1989, 9:4119-4130.

10.

COLEMANjE: The Role of Zn(ll) in RNA and DNA Polymerases. In Zinc EnzFmes, Edited by Spiro TG. New York: John Wiley & Sons; 1983:221-252.

11. TREtCHI, RrVAM, SENTENACA: Zinc-binding Subunits of Yeast ,. RNA Polymerases. J Biol Cbem 1991, 266:21971-21976. First direct evidence for the binding of zinc by specific cysteine residues in the second largest subunit of RNA polymerases. 12. •

BERGSt.AND KJ, HASEtKORN R: Evolutionary Relationships a m o n g Eubacteria, Cyanobacteria, and Chloroplasts: Evidence from the rpoC1 G e n e of A n a b a e n a sp. Strain PCC 7120. J Bacteriol 1991, 173:3446-3455. The authors show that a split in the largest subunit of RNA polymerase occurs in both cyanobacteria and chloroplasts. The break-point of this split is located at a different position from that occurring in archaebacteria. 13. •

PAI.ENIKB, HASELKORN R: Multiple Evolutionary Origins of Prochlorophytes, the Chlorophyll b-Containing Prokaryotes. Nature 1992, 355:265-267. An example of the use of conserved regions in RNA polymerases, together with PCR, to generate sequence data for answering an evolutionary question: the origin of the prochlorophytes and their relationship to the endosymbiotic ancestor of green chloroplasts.

ARCHAMBAULTJ, DREBOT /VIA, STONE JC, FRIESEN JD: Isolation and Phenotypic Analysis of Conditional-lethal, Linker-insertion Mutations in the Gene Encoding t h e Largest Subunit of RNA Polymerase II in S a c c h a r o m y c e s cerevisiae. Mol Gen Genet 1992, 232:408--414. This paper presents a systematic investigation of the effects of insertion mutations in the largest subunit of RNA polymerase and the resultant phenotypes. 14. **

15, •

PETEIZSEN SK, HANSEN FG: A Missense Mutation in the rpoC Gene Affects Chromosomal Replication Control in Escherichia coll. J Bacteriol 1991, 173:5200-5206. The authors investigate the vital, but still to be sufl~ciendy elucidated, role of RNA polymerase in the control of chromosomal copy number. 16.

BARTOLOMEIMS, CORDEN JL: Localization of an ct-amanitin Resistance Mutation in the G e n e Encoding the Largest Subunit of Mouse RNA Polymerase II. Mol Cell Biol 1987, 7:586-594.

17.

MARTINC, OKAMURAS, YOUNG R: Genetic Exploration of interactive Domains in RNA Polymerase II Subunits. Mol Cell Biol 1990, 10:1908-1914.

18. ,.

YANO R, NOMURA M: Suppressor Analysis of Temperaturesensitive Mutations of the Largest Subunit of RNA Polymerase I in Saccharomyces cerevisiae a Suppressor Gene Encodes the Second-largest Subunit of RNA Polymerase I. Mol Cell Biol 1991, 11:754-764. These atnhors use genetics in a first attempt to generate a three dimensional picture of the subunit interactions of RNA polymerase. 19. •

LIAO S-M, TAYLOR ICA, KINGSTON RE, YOUNG RA: RNA Polymerase II Carboxy-terminal Domain Contributes to the Response to Multiple Acidic Activators In Vitro. Genes Dev 1991, 5:2431-2440. This paper investigates the role of the carboxT-temlinal clomain of RNA pol II in transcriptional activation. (Also see [20•1.) 20. •

PETER.SONCL, KRUGERW, HERSKOWI'VZI: A Functional lnteraction Between the C-terminal Domain of RNA Polymerase II and the Negative Regulator SINI. Cell 1991, 64:1135-1143. Provides a further analysis (see [19 • ] ) of the functions of the carbo:,q,terminal domain of the largest subunit of pol If. 21.

S\xq?.E-FSERD. NONET M, YOUNG RA: Prokaryotic and Eukaryotic RNA Polymerases have Homologous Core Subunits. Proc Natl Acad Sci USA 1987, 84:1192-1196.

22. •

SPARKOWSKIJ,. DIns A: Simultaneous Gain and Loss of Functions Caused by a Single Amino Acid Substitution in the subunit of Escherichia coli RNA Polymerase: Suppression of nusA and rho Mutations and Conditional Lethality. Geneti~ 1992, 130:411-428. Describes a mutation in tpoB (occurring in region 6 of the E. coli 13 subunit) that "affects termination. 23. •

ROCKWELLP, GOTTESMANME: An Escherichia coli rpoB Mutation that Inhibits Transcription of Catabolite-sensitive Operons. J Mol Biol 1991, 222:189-196. This work characterizes a mutation in rpoB(occurring in region 8 of the E coil ~ subunit) that may affect promoter clearance of transcription elongation. 24.

LEE J, KASHI_EV M, BORUKHOV S, GOmFAaB A: A 13 Subunit Mutation Disrupting the Catalytic Function of Escherichia coli RNA Polymerase. Proc Natl Acad Sci USA 1991, 88:6018~5022.

25. •

MUSTAEV A, KASHLEV M, LEE J, POLYAKOV A, LEBEDEV A, ZAI.E.NSKAYAK, GRACHEV M, GOLDFARB A, NIKIFOROVV: Mapping of the Priming Substrate Contacts in the Active Center of Escherichia coli RNA Polymerase. J Biol Cbem 1991, 266:23927-23931. These authors characterize mutations near the catalytic site of RNA polymerase. 26.

JzN DJ, GROSS CA: Mapping and Sequencing of Mutations in the Eschericbia coli rpoB G e n e that Lead to Rifampicin Resistance. J Mol Biol 1988, 202:45-58.

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BORUKHOVS, SEVERINOV K, KASHLEV M, LEBEDEV A, BA.'CS I, ROWLANDGC, LIM P-P, GLASS RE, NIKIFOROV V, GOLDFARB A: Mapping of Trypsin Cleavage and Antibody-binding Sites and Delineation of a Dispensable Domain in the Subunit of Escherichia colt RNA polymerase. J Biol CIJem 1991, 266:23921-23926. Demonstrates that a region of the 13 subunit present in eubacteria, but not chloroplasts, is not required for polymerase function. This region could be of particular use in phylogenetic studies. •.

28. •

SEVERINOVK, MUSTAEVA, KASHLEVM, BORUKHOVS, NIKIFOROV V, GOt.OFARB A: Dissection of the 13 Subunit in the Escherichia colt RNA Polymerase into Domains by Proteolytic Cleavage..l Biol Cl.~em 1992, 267:12813-12819. This work is part of a series. See also Bomkhov et al. [27•*]. 29. SHIRAIT, GO M: RNase-like Domain in DNA-directed RNA T Polymerase 11. Proc Natl Acad Sci USA 1991, 88:9056--9060. his paper points out an interesting similarity between RNases and eukaryotic DNA-dependent RNA pol~uerases, but the relevance of this similarity is ambiguous. 30.

IGARASHIK, FUJITA N, ISHIHAMAA: Sequence Analysis of two Temperature-sensitive Mutations in the Alpha Subunit Gene (rpoA) of Escberichia coil RNA Polymerase. Nucleic Acids Res 1990, 20:5945-5948.

31. o.

DEQUARD-CH,mL.\'rM, RaX,AM, CARLES C, SENTENAC A. RPCI9, the Gene for a Subunit C o m m o n to Yeast RNA Polymerases A(1) and C(III)..I Biol Cbem 1991, 266:15300-15307. Demonstrates a specific relationship between subunits of eukaryotic polymerases and the cx subunit of prokaryotic pol}~nerase.

32. ,,•

THOMuXSMS, GI&sS RE: Escherichia coil rpoA Mutation w h i c h Impairs Transcription of Positively Regulated Systems. Mol Microbiol 1991, 5:2719-2725. One of a recent group of papers investigating mutations affecting the functioning of ~poA, which encodes the least understood RNA polymerase subunit (see [33••-35 *•]). 33. ••

IGAI~'6HI K, HAN&MURAA, MAKINO K, AIBA H, /VI~tlNO T, NAKATAA, ISHIHAMAA: Functional Map of the ~x Subunit of Escherichia coli RNA Polymerase: T w o Modes of Transcription Activation by Positive Factors. Proc Natl Acad Sci USA 1991, 88:8958--8962. One of a recent group of papers investigating mutations ,affecting the functioning of rpoA (see [32"*,34"',35"*] ). 34. ••

LOMBAREK)M-J, BAGGA D, Mna.ER CG: Mutations in tpoA Affect Expression of Anaerobically Regulated G e n e s in Salmonella typhDnurium. J Bacteriol 1991, 173:7511-7518. One of a recent group of papers im,estigating mutations affecting the functioning of rpoA (see [32•%33°%35 °°] ). 35.

IGtUtASHIK, ISHII-L.'~\IAA: Bipartite Functional Map of the E. colt RNA Polymerase cx Subunit: Involvement of the C-terminal Region in Transcription Activation by cAMP-CRP. Cell 1991, 65:1015-1022. One of a recent group of papers investigating mutations affecting the functioning of IpoA (see[32"•-34** ] ). • •

36.

HELblANNJD, CHAblBERUNMJ: Structure and Function of Bacterial Sigma Factors. A n n u Re\, Biocbem 1988, 57:839-872.

37. •

LOr,'ETTO M, GVaBSKOV M, GROSS CA:The Sigma 70 Family: Sequence Conservation and Evolutionary Relationships. J Bacteriol 1992, 174:3843-3849. This work details valuable update of ~ factor alignments, which is also used to generate a gene tree for the evolution of multiple ~ factors. 38. *

MAIJKS, HISATAKEK, SUMIMOTO H, HORIKOSHI M, ROEDER RG: Sequence of General Transcription Factor TFIIB and Relationships to O t h e r Initiation Factors. Proc Natl Acad Sci USA 1991, 88:9553-9557. This work documents relationships between bacterial ~ factors and eukaryotic transcription factors (also see [39",40"]). 39. *

A recent paper (also see [38 •,40 • ]) documenting relationships between bacterial cr factors and eukar3,otic transcription factors. 40.

SUMIMOTO H, OHKUMA Y, SINN E, KATO H, SHIMASAKI S, HOPdKOSHIM, ROEDER RG: Conserved Sequence Motifs in the Small Subunit of H u m a n General Transcription Factor TFIIE. Nature 1991, 354:401-404. A recent paper (also see [38",39"] ) documenting relationships between bacterial ~ factors and eukaryotic transcription factors. •

JANG SH, JAEHNING JA: T h e Yeast Mitochondrial RNA Polymerase Specificity Factor, MTFI, is Similar to Bacterial Sigma Factors. J Biol Cbem 1991, 266:22671-22677. These authors present elucidating results that are facilitating a better understanding of the very composite nature of eukary,otic organisms. 41.

••

42.

MEMETS, SAIIRIN W, SENTENAC A: RNA Polymerases B and C are More Closely Related to Each O t h e r than to RNA Polymerase A. J Biol Chem 1988, 263:10048-10051.

43.

POHLERG, LEFFEIt¢, H, GROPP F, PAhM P, KLENKH-P, LO'I'I.'SI'EICH F, GARREt" RA, ZIUaG W: &rchaebacterial DNA-dependent RNA Polymerases Testify to the Evolution of the Eukaryotic Nuclear G e n o m e . Proc Natl Acad Sci USA 1989, 86:4569-4573.

44.

R r ~ , MC, L~KE JA: Evidence that EukarTotes and Eocyte Prokaryotes are Immediate Relatives. Science 1992, 257:74-76.

45.

SEIFARTH W, PE'I'ERSEN G, KONTERMANN R, R~'A M, HtlET of the G e n e s Coding for the Second-largest Subunits of RNA Polymerases I and III of Drosophila melanogaster Mol Gen Genet 1991, 228:424--432.

J, 13AUTZ EKF: Identification

46.

J,~IES P, WHEI.EN S, HALL BD: The RET1 Gene of Yeast Encodes t h e Second-largest Subunit of RNA Polymerase 111.J Biol Chem 1991, 266:5616-5624.

47. ••

ROHE M, SCHRUENDERJ, TUDZYNSKI P, MEINHARDT F: Phylogenetic Relationships of Linear, Protein-primed Replicating Genomes. Curr Genet 1992, 21:173-176. These authors use both DNA and RNA-polymerase gene sequences to study the origin of linear plasmids. 48.

KLENKl I-P, PAh\I P, LOTTSPEICH F, ZIIJJG W: C o m p o n e n t H of the DNA-dependent RNA Polymerases of Archaea is Homologous to a Subunit Shared by the Three Eucaryal Nuclear RNA Polymerases. Proc AYftl Acad Sci USA 1992, 89:407-410.

49. •

STEC DS, HILl. MGlll, COLLINS PL: Sequence Analysis of tbe Polymerase L Gene of H u m a n Respirator), Syncytial Virus and Predicted Phylogeny of N o n s e g m e n t e d Negative-strand Viruses. I'irolo$o, 1991, 183:273-287. These attthors use highly conserved viral polymerase gene sequences to explore viral phylogenies. 50. •

KOONJNEV: T h e Phylogeny of RNA-dependent RNA Polymerases of Positive-strand RNA Viruses. J Gen I/irol 1991, 72:2197-2206. A recent example o f the use of polymerase sequence data in developing viral phylogenetic trees. 51. .

RJB,vsJC, WtCKNERRB: RNA-dependent RNA Polymerasc Cons e n s u s Sequence of the L-A Double-stranded RNA Virus: Definition of Essential Domains. Proc Natl Acad Sci USA 1992, 89:2185-2189. In this work, the evolutionary diversity of double-stranded RNA x~mses is used to define essential domains of their RNA-dependent RNA polymerase. 52.

TAYLOR EW, JAAKKOLA J: A Transpositon of the Reverse Transcriptase Gene Reveals U n e x p e c t e d Structural Homology to E. colt DNA Polymerase I. Genetica 1991, 84:77~6.

53.

PISANIFM, DE M~rnNo C, Ross~ M: A DNA Polymerase from the Archaeon Sulfolobus Solfataricus Shows Sequence Simi. larity to Family B DNA Polymerases. Nucleic Acids Res 1992, 20:2711-2716.

OHKUMAY, SUNIIMOTOH, HOFFMANNA, SHIMASAKIS, HORIKOSHI M, ROEDER RG: Structural Motifs and Potential Sigma Homo-

logies in the Large Subunit of H u m a n General Transcription Factor TFIIE. Nature 1991, 354:398--401.

B Palenik, Marine Biology, The Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093-0202, USA.