Orthologs of the Small RPB8 Subunit of the Eukaryotic RNA Polymerases

Biology Direct BioMed Central

Discovery notes Open Access Orthologs of the small RPB8 subunit of the eukaryotic RNA polymerases are conserved in hyperthermophilic Crenarchaeota and "Korarchaeota" Eugene V Koonin*1, Kira S Makarova1 and James G Elkins2

Address: 1National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA and 2Microbial Ecology and Physiology Group, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA Email: Eugene V Koonin* - [email protected]; Kira S Makarova - [email protected]; James G Elkins - [email protected] * Corresponding author

Published: 14 December 2007 Received: 13 December 2007 Accepted: 14 December 2007 Biology Direct 2007, 2:38 doi:10.1186/1745-6150-2-38 This article is available from: http://www.biology-direct.com/content/2/1/38 © 2007 Koonin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract : Although most of the key components of the transcription apparatus, and in particular, RNA polymerase (RNAP) subunits, are conserved between archaea and eukaryotes, no archaeal homologs of the small RPB8 subunit of eukaryotic RNAP have been detected. We report that orthologs of RPB8 are encoded in all sequenced genomes of hyperthermophilic Crenarchaeota and a recently sequenced "korarchaeal" genome, but not in Euryarchaeota or the mesophilic crenarchaeon Cenarchaeum symbiosum. These findings suggest that all 12 core subunits of eukaryotic RNAPs were already present in the last common ancestor of the extant archaea. Open peer review: This article was reviewed by Purificacion Lopez-Garcia and Chris Ponting.

Findings structure, Rpb8 interacts with the so-called pore module The core components of the information-processing sys- of Rpb1 at a defined motif that is conserved in both tems, and in particular, the transcription machinery, are eukaryotic and archaeal Rpb1 orthologs [8]. In addition, conserved between archaea and eukaryotes, and distinct Rpb8 genetically interacts with another small subunit, from the bacterial versions. The heteromultimeric eukary- Rpb6, that is also adjacent to the pore module in the core otic RNAPs consist of 12 subunits (Rpb1–12), of which 11 RNAP structure [6]. It has been suggested that Rpb8 and are conserved in archaea and eukaryotes whereas one, Rpb6, together with the pore module, form a distinct Rpb8, is thought to be unique for eukaryotes [1-6]. Rpb8 functional unit [6]. The exact role of the small subunits is a small protein that typically consists of ~120–150 remains unknown although both Rpb6 and Rpb8 are con- amino acids and shows relatively poor sequence conserva- served in all eukaryotes, are shared between RNAP I, II tion in eukaryotes. The structure of Rpb8 has been solved, and III, and are essential for yeast growth. Regardless of originally, by solution NMR [7] and, subsequently, as part the precise function of Rpb8, the purported absence of of the RNAP II core, by X ray crystallography [8]. The two this RNAP subunit ortholog in archaea is the major gap in structures are in good agreement and indicate that Rpb8 the picture of the otherwise exact correspondence forms a distinct version of the OB(oligonucleotide-oli- between the core transcriptional machineries of eukaryo- gosaccharide-binding) fold [9] that is characterized by a tes and archaea, and is highly unexpected considering the distinct pattern of 9 β-strands and a pair of invariant gly- conservation of all other subunits including the func- cines in the turn between strands 7 and 8. In the RNAP II tional partner of Rpb8, Rpb6.

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In the course of the genome annotation for the first ophilic crenarchaeon for which the genome sequence is sequenced member of the "Korarchaeota", a putative deep currently available) do not. branch of archaea ([10] and manuscript in preparation), one of us (JGE) identified a short (110 amino acids) pre- In retrospect, we became aware that the protein we identi- dicted protein for which some of the best hits in a BLAST fied as the crenarchaeal ortholog of Rpb8 has already been search [11] were the eukaryotic Rpb8 subunits. Although described as one of the 13 experimentally defined subu- the sequence similarity was not statistically significant, nits of the RNAP of the crenarchaeon Sulfolobus acido- this observation prompted a systematic search for possi- caldarius and designated RpoG [12], and the ortholog ble archaeal homologs of Rpb8. BLAST searches started encoded in the genome of S. solfataricus has been accord- with the sequences of Rpb8 subunits of various eukaryotic ingly annotated [13]. Given these data, it appears sensible species, again, showed statistically not significant similar- to adopt the designation RpoG for the archaeal orthologs ity to a distinct set of crenarchaeal proteins that were sim- of Rpb8. In a subsequent global analysis of mRNA stabil- ilar in size to (or slightly shorter than) Rpb8. However, ity in the two Sulfolobus species, it has been shown that the reciprocal iterative PSI-BLAST searches (inclusion E-value RpoG mRNA is markedly more stable than mRNAs of cut-off 0.01) started with some of these crenarcheal other RNAP subunits, and the apparent uniqueness of this sequences showed significant similarity to Rpb8. For subunit in Sulfolobus has been emphasized [14]. The example, a search with the sequence from Hyperthermus present analysis clarifies the situation by showing that butylicus (Hbut_0467) used as the query retrieved the RpoG is conserved throughout the hyperthermophilic Rpb8 sequence from fission yeast Schizosaccharomyces Crenarchaeota (and at least one korarchaeote). pombe in the 2nd iteration, with a E-value of 0.004, and numerous eukaryotic Rpb8 sequences in the 3rd iteration, Although the small size of Rpb8 and its archaeal orthologs with highly significant E-values. Similarly, a search with (RpoG) hampers reliable phylogenetic analysis, the maxi- the sequence from Igniococcus hospitalis (Igni_1165) mum likelihood tree we constructed shows a clear separa- retrieved Rpb8 from Entamoeba histolytica in the 5th iter- tion of the eukaryotic and crenarchaeal branches, and ation, with an E-value of 4 × 10-5). within the latter, the split between Thermoproteales and Sulfolobales (Fig. 1b). Interestingly, the korarchaeal Examination of a multiple alignment of the Rpb8 RpoG clustered with Thermoproteales in a strongly sup- sequences from diverse eukaryotes and the putative ported branch (Fig. 1b). Broader implications of this archaeal counterparts showed remarkable conservation of observation for the evolution of the "Korarchaeota" all elements of the OB-fold, in particular, the diagnostic remain to be investigated. motif between strands 7 and 8 (although one of the two glycines that are invariant in Rpb8 is replaced in a subset In the archaeal genomes, the rpoG gene is embedded in a of the putative archaeal homologs (Fig. 1a). Furthermore, notable, partially conserved genomic context (Fig. 1b). secondary structure prediction for the archaeal proteins With few exceptions, rpoG forms either a codirectional or showed a near-perfect superposition with the secondary a divergent but potentially coregulated gene pair with a structure elements extracted from the crystal structure of gene encoding a small RNA-binding protein (COG1958) Rpb8 [8] (Fig. 1a). Taken together, these findings indicate that is orthologous to eukaryotic Lsm6 and is implicated that the detected small archaeal proteins are bona fide in RNA-processing. In most of the Sulfolobales, the latter homologs of Rpb8. gene is adjacent to a gene for a tRNA modification enzyme, queuine/archaeosine tRNA-ribosyltransferase. Small proteins homologous to Rpb8 were identified in all Another common neighbor of rpoG is the gene for tran- 10 sequenced genomes of hyperthermophilic Crenarchae- scription elongation factor TFIIIB, with a divergent orien- ota and the only available korarchaeal genome. Each of tation in the majority of Thermoproteales and a these genomes encodes a single Rpb8 homolog which codirectional orientation in the korarchaeote. Finally, mimics the situation in eukaryotes where no paralogs of Thermofilum pendens appears to have a more complex Rpb8 are detectable. By contrast, n homologs of these pro- operon organization, with the genes for another RNAP teins were identified in Euryarchaeota and the mesophilic subunit, a transcription factor and a ribosomal protein in crenarchaeon Cenarchaeum symbiosum, despite extensive the same predicted operon with rpoG (Fig. 1b). This search including running a position-specific scoring genomic context suggests that, in Crenarchaeota, RpoG is matrix for Rpb8 and their crenarchaeal-korachaeal likely to be involved in a tight functional cooperation homologs against a dedicated database of euryarchaeal with TFIIIB, and could also contribute to coupling tran- and C. symbiosum protein sequences. Thus, we conclude scription with RNA processing and modification. that all thermophilic Crenarchaeota and at least one korarchaeote encode a single ortholog of eukaryotic Rpb8 The finding described here fills the last gap in the one-to- whereas Euryarchaeota and C. symbiosum (the only mes- one correspondence between the RNAP subunits of

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A 126353193 C.maquilingensis 4 YRGTYKVTGIEYE----PNNVVKLRLEGEGSL-ITVELPAI--INKFKQGDSVLISLSSS 2 ENYKGNWDVYMWGMVYY 0 LGENFMRISIGGFILHLEGNVS-----SRPNLGDKIYVGLK 111 119719267 T.pendens 27 RLMRGVVRSVGRLGYALVERVGIAGDGVE----VEVEIPVR4AGLGLREGVEVVFEVARE 0 AGSIDEWSVVMQGEVYL 2 EGESAVYASLGGLQLVVKSPKIY----NEFNVGDKVYFKLA 141 145592327 P.arsenaticum 1 MEFPLRVVEISDG----LPNVIKLRDGVE----VTVELPLEKIGVKLRVGDQVKLVIHKE 1 DSDLTKYKIYAWGVVYF 0 VGKDMTRISIGGLQLDIGTE------LPLKVGEKVYIGIL 104 18313112 P.aerophilum 1 MEFSLAVVDVTND----MPNVIKLKGDVE----ATIELPLEKLGVVFSVGDRLKLVLQKE 1 DQDLNKYKVYAWGIVYY 0 VGNGMTRISIGGLQLDIMKE------LPLKAGEKIYVGIL 104 119872466 P.islandicum 1 MEFLLKVEEISND----FPNTIKLKGDVD----ITIELPLNILGIKLSKGENVKLVLQQE 1 DKELHKYKVYAWGIVYY 0 VGNNITRISIGGLQLDIMKE------LPLKVGEKVYIGII 104 126459669 P.calidifontis 1 MEFSLSVVDITSD----FPNVIKLSGDVE----ATVELPLEKLGVALKKGDKIRLVLSKE 1 DGDLSKYKVYMWGIVYH 0 VAEELTRISIGGLQLDIKKK------LPLAIGEKVYIGIL 104 118431688 A.pernix 3 KKLDLKIESMEPGK---LQGQLIATAKSKK---ATVMFDVIKDLVPLSEGDRIVIEVLEE 1 PKSLDKYVFCGHGYKVP 1 EKSGVDILSVWGILFVFEPQ------IELELDKKYYVCIS 109 124027356 H.butylicus 7 VAFEGTVVSVEPEL---IPKLYVTRIKSNNGE-YEVEMDTHSELIVFKEGDNVLVELSRS 1 PEYRDGVDFVGRGTVVS 4 GETYAALISMGGLLFIIRSRKE-----LDLAPVEKVYVKVA 120 15897220 S.solfataricus 9 IILSCEINSIERGS---LKNLSMVNMSCNG---FNVSFDIIDSINIFSQKEKVKVIISKN 1 PSYSHD-DFCGHGYIVT 9 GNKYTTIISLYGLLVKIISNKESFLRTSQLNIMDHVYFCVK 129 70606468 S.acidocaldarius 5 MQGTCKISSIEKGA---LKNLYVVKMDCDND--LKIEFDITKELSIFSKDEEVTFIISRE 1 PEYSEK-DFCAHGYLFL 5 DGSFIDEISLYGLIVKILSKNGLI-NSKLFKMMDHVYYCVK 121 15920524 S.tokodaii 9 VETTCKITSIDKGS---LKGLIIIKMDCGI---YQVEFDILESINIFKQEENVSMIISRE 1 PVYTSN-DFCAHGYLFY 4 GNKILDQISLFGLIVKIYSEKGLI-GNSLFKMMDHVYFCVK 123 126465807 S.marinus 6 VSYECKVRSIEPLR---YIGMYRAISDCDS---VEITLEMNEKLMKISEGDDLVINISTS 0 KKECLQHHFCGKGHVVS 4 DNTYRVVVSIAGILVVIKMKEQPK---SPFKVMNELYIGVS 118 146305016 M.sedula 7 FSEMCKIDSIKKGE---LRDLLIVAVRCQD---TEFNLDMISSLNMFKDGENVNVILSKE 0 RPDFTEKDFCAHGYVVT 5 DGTFVTIISFFGPLLRISSKDDFL-KRAGLNVMDHVYFCVR 122 156937953 I.hospitalis 1 MECSGKVTDIQKGK---IPYTNILVFECDD---KKVEMLLHEDLITFVKGEAARFVVSEE 1 PQFKQGVDFCGRGVLVR 0 DDGDKKLFSIGGFVVVLHNFQE------KFKENVKYYICLM 107 Korarchaeum sp. 11 YLSKVRLSSVRSDP---IFDINILSFESLDRS-FEATLEFPKGLIDLKEGMEADLTLGEE 0 ----GEGDIIMKGVVYK 2 DSSRTIEISFHGLLMKLTYSKAAP---FKLSQGEEVYLTIK 119 154422638 T.vaginalis 4 LTKNFRCTAVDPDGKQ-FDDVSRIKATTDEGD-YELVLDYNCFIYEIKQDQLFQLNISTQ 20 STLAASYEYIMHGKVYN 6 TNRATVYVSYGGLLMSLTGDRNTI---AGIQRGKEVYLLIS 142 71075425 G.lamblia 4 YSSRFSIASFDPECVKYYDNIGRVLLESTDDPDVFLTLDYNLELYPKLNKN5VQILSSIS 21 HDAYPDLDYAMHGTVFE 4 GDKVDMFISFGGLIAKLQAPAFRL---ERFKFGMKLYLLCC 147 84999542 T.annulata 8 FEDRFLVRSIDNSK---FERVSRLSAKSTGFD-AELLLDYNSDLLQVYNKQVLHILITNS 16 PSLLGDYEYAMYGKIFK 5 SETRTIYASFGGLLMSLTADKQVV---ADLDLDMKIYLLVR 139 146091778 L.infantum 8 LEDTFTVNGVNTEGTV-YLRVSRIRCVNQDGS-LTITTDINSEEFPVSTNDRLTIVLANS 19 ETRLNDCDYAMHGRVYG 6 GLEVNVHISCGGLLVNIVGKPQGL---RDVHYNSDVYILIK 145 118345758 T.thermophila 8 LEDRFKLEEIKKEEK--FSKVERVVLVSDNN--IKIEIDIHTDLYSMKKGNQYWVMITSS 18 ESIKDKFEYVMHGKIYN 6 AKTVSIFASFGGLLMKVSGKDEDL---KNFNVDSRIYILIR 142 145527078 P.tetraurelia 9 IDEYFKVEDIDQAGQS-FKRVSRIKCKSKSGE-VDMELDVNIDIYPIDKGSEYRVLIVRS 20 NELLEKFQYAMFGRVFK 5 NKMINVFASFGGLILKLSGKSEEL---DKFADDQRVYLLMR 146 66362188 C.parvum 8 FEDRFVINSVDSGK---FDRIGRIRGRSVGFD-ADLILDINNELFPVREKESLYIGLSSQ 15 QTLMDQYDYVMYGKIYR 5 SDRRSLYASFGGLLMSLTADKNLV---GDFSLDMRLYCLVR 138 124805803 P.falciparum 7 FEDRFVISSVDNSK---FEKVSRIKAKSTGYD-AELILDVHSELFKVEEKKAIYLALQDK 14 NVPLNNIEYIMSGRIFK 5 SERRTVYASFGGLMMALTTDKQFI---GDLEIDMKIYLLTK 136 133340 S.cerevisiae 6 FDDIFQVSEVDPGR---YNKVCRIEAASTTQDQCKLTLDINVELFPVAAQDSLTVTIASS 24 RSLADDYDYVMYGTAYK 5 KDLIAVYYSFGGLLMRLEGNYRNL---NNLK-QENAYLLIR 145 108712076 O.sativa 6 FEDIFTVTRLDPDGKK-FDRVSRIEARSDQFD-MYMQLDVATDVYPMHPGDRFTMVLVPT 17 KTLADKYEYVMHGKLYK 11 AKKVEMYASFGGLLVMLKGDPSSA---ANFELDQRLFLLMR 146 28466897 A.thaliana 8 FEDIFVVDQLDPDGKK-FDKVTRVQATSHNLE-MFMHLDVNTEVYPLAVGDKFTLALAPT 17 KTLADKYEYIMHGKLYK 7 TPKAELYVSFGGLLMLLKGDPAHI---SHFELDQRLFLLMR 144 66816149 D.discoideum 6 FEDTFEVKEIDPDGKK-FDRVSRFVCYSENYE-MDLQIDIATHIYPLGHE-RFKMVLASS 17 PSLADKYDYVMYGKIFK 6 SSKVSVFASFGGLLVLLQGDPRYL---PGIELDARIYLLIK 140 2833302 C.elegans 6 FDDMFKVKSVDPDGKK-FDRVSRYFCDAESFK-MELIIDINSQIYPLKQNDKVRLVLATT 17 YPRIKQYEYVMYGKVYR 9 GGKLAAYASFGGLLMRLKGEAINL---HGFEVDMNLYLLMK 144 21358365 D.melanogaster 6 FEDIFNVKDMDPEGKK-FDRVSRLHCESESFK-MDLILDINSWLYPMELGDKFRLVLATT 18 GTRADSFEYVMYGKIYR 9 ASRLSAYVSFGGLLMRLQGDANNL---HGFEVDQHMYLLMK 145 99032143 pdb:2F3I(H.sapiens)6 FEDIFDVKDIDPEGKK-FDRVSRLHCESESFK-MDLILDVNIQIYPVDLGDKFRLVIAST 18 PSRADQFEYVMYGKVYR 10 ATRLSAYVSYGGLLMRLQGDANNL---HGFEVDSRVYLLMK 146 consensus/95% h...h.h..hp...... h..h.hh...s.....h.h.h.....h..h.....h.h.h..p...... phhh.G.hh...... hShhGh.h.h...... h....phhhhh Secondary str. (pdb:2F3I) EEEEEEEEEE EEEEEEEE EEEEEE EEEE EEE EEEE EEEEEEE EEEEEEE HHHH EEE PSIPRED Secondary str. EEEEEEEEEE EEEEEEEE EEEEEEE EEEE EEEEEEEE EEEEEEEEE EEEEEEE EEEEEEEE HHH EEEEEE B

Trichomonas vaginalis 154422638 57 Giardia lamblia 71075425 Theileria annulata 84999542 50 Cryptosporidium parvum 66362188 Plasmodium falciparum 124805803 Saccharomyces cerevisiae 133340 Leishmania infantum 146091778 Tetrahymena thermophila 118345758 55 Paramecium tetraurelia 145527078 Dictyostelium discoideum 66816149 Caenorhabditis elegans 2833302 Drosophila melanogaster 21358365 71 Homo sapiens 99032143 Oryza sativa 108712076 99 Arabidopsis thaliana 28466897 Aeropyrum pernix 118431688 84 Staphylothermus marinus 126465807 Hyperthermus butylicus 124027356 91 Ignicoccus hospitalis 156937953 Sulfolobus solfataricus 15897220 78 Metallosphaera sedula 146305016 Sulfolobus acidocaldarius 70606468 Sulfolobus tokodaii 15920524 50 Thermofilum pendens 119719267 *** Korarchaeum sp. 55 Caldivirga maquilingensis 126353193 90 Pyrobaculum arsenaticum 145592327 Pyrobaculum aerophilum 18313112 100 Pyrobaculum islandicum 119872466 50 Pyrobaculum calidifontis 126459669

DNA-directed RNA polymerase subunit RpoG DNA-directed RNA polymerase subunit RpoF Small RNA-binding protein, ortholog of LSM6 Uncharacterized protein, specific for Thermoproteales Transcription initiation factor TFIIIB, Brf1 subunit Ribosomal protein S17e Queuine/archaeosine tRNA-ribosyltransferase Transcription factor CBF/NF-Y histone

OrthologsFigure 1 of Rpb8 in archaea Orthologs of Rpb8 in archaea. (a) Multiple alignment of eukaryotic Rpb8 subunits and their archaeal orthologs (RpoG). The alignment was constructed using the combination of the results obtained with PROMALS [15] and MUSCLE [16], followed by manual correction on the basis of secondary structure prediction that was obtained using PSIPRED [17] and local alignments generated by PSI-BLAST. Sequences are denoted by their numeric Genbank Identifiers (GI numbers) and species names. The full species names are given in Figure 2. The positions of the first and the last residues of the aligned region in the corresponding protein are indicated for each sequence. The numbers within the alignment represent poorly conserved inserts that are not shown. The numbers of omitted amino acids for T. pendens and G. lamblia are indicated by reverse shading. Positions with identical amino acids in all aligned sequences are in bold face. The coloring is based on the consensus shown underneath the alignment; 'h' indicates hydrophobic residues (ACFILMVWYH), 'p' indicates polar residues (STEDKRNQH), 's' indicates small residues (AGCVDS). Secondary structure is shown for the crystal structure of human Rpb8 (pdb 2F3I); 'H' indicates α-helix and 'E' indicates extended conformation (β-strand). The PSIPRED secondary structure prediction is shown underneath the experimental secondary structure. The glycine doublet that is invariant in eukaryotic Rpb8 sequences is boxed. (b) Phylogenetic and genomic contexts of Rpb8/RpoG. The maximum likelihood phylogenetic tree of Rpb8/RpoG was constructed by local rearrangement of an original minimum evolution (Fitch) tree [18] using the MOLPHY program [19]. MOLPHY was also used to compute RELL bootstrap probabilities, which are indicated (as percentages) for selected major branches. Each terminal node of the tree is labeled by the full species name and the GI number. The genomic neighborhoods of the rpoG gene in Crenarchaeota and the "korarchaeal" genome are shown to the right of the respective branches of the tree. Ortholo- gous genes are shown by arrows of the same color.

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archaea and eukaryotes, with the implication that the homolog among the "korarchaeal" proteins; all authors archaeal "parent" of eukaryotes already possessed the read, edited and approved the final version of the manu- intricate 12-subunit organization of RNAP. Surprisingly, script. however, Euryarchaeota and the only available genome of a mesophilic crenarchaeon appear to lack an ortholog of Acknowledgements Rpb8, a conclusion that is compatible with the report on EVK and KSM are supported by the Intramural Research Program of the the reconstruction of a fully active RNAP of the euryar- National Institutes of Health, National Library of Medicine. JGE acknowl- chaeon Methanocaldococcus jannaschii from 12 recom- edges the support of korarchaeal genome sequencing by the Joint Genome binant proteins which, obviously, did not include Rpb8 Institute Community Sequencing Program. [1]. Depending on the adopted evolutionary scenario, it is References conceivable that Rpb8 emerged in the crenarchaeal line- 1. Ouhammouch M, Werner F, Weinzierl RO, Geiduschek EP: A fully age or, perhaps, more plausibly, that it was already present recombinant system for activator-dependent archaeal tran- in the common ancestor of all extant archaea but lost at scription. J Biol Chem 2004, 279(50):51719-51721. 2. Werner F, Eloranta JJ, Weinzierl RO: Archaeal RNA polymerase the base of the euryarchaeal branch. Regardless of the subunits F and P are bona fide homologs of eukaryotic RPB4 solution to this conundrum, experimental study of func- and RPB12. Nucleic Acids Res 2000, 28(21):4299-4305. tional differences between RNAPs of Euryarchaeota and 3. Werner F, Weinzierl RO: A recombinant RNA polymerase II- like enzyme capable of promoter-specific transcription. Mol Crenarchaeota should be illuminating, given the unusual Cell 2002, 10(3):635-646. difference in their predicted subunit composition. 4. Langer D, Hain J, Thuriaux P, Zillig W: Transcription in archaea: similarity to that in eucarya. Proc Natl Acad Sci U S A 1995, 92(13):5768-5772. Abbreviations 5. Zaros C, Briand JF, Boulard Y, Labarre-Mariotte S, Garcia-Lopez MC, RNAP: RNA polymerase. Thuriaux P, Navarro F: Functional organization of the Rpb5 subunit shared by the three yeast RNA polymerases. Nucleic Acids Res 2007, 35(2):634-647. Competing interests 6. Briand JF, Navarro F, Rematier P, Boschiero C, Labarre S, Werner M, Shpakovski GV, Thuriaux P: Partners of Rpb8p, a small subunit The author(s) declare that they have no competing inter- shared by yeast RNA polymerases I, II and III. Mol Cell Biol ests. 2001, 21(17):6056-6065. 7. Krapp S, Kelly G, Reischl J, Weinzierl RO, Matthews S: Eukaryotic RNA polymerase subunit RPB8 is a new relative of the OB Reviewers' reports family. Nat Struct Biol 1998, 5(2):110-114. Reviewer 1: Purificacion Lopez-Garcia, Universite Paris- 8. Cramer P, Bushnell DA, Kornberg RD: Structural basis of tran- Sud scription: RNA polymerase II at 2.8 angstrom resolution. Sci- ence 2001, 292(5523):1863-1876. 9. Murzin AG: OB(oligonucleotide/oligosaccharide binding)-fold: This manuscript reports the identification of genes orthol- common structural and functional solution for non-homologous sequences. Embo J 1993, 12(3):861-867. ogous to RPB8 in archaea. This observation is very inter- 10. Barns SM, Delwiche CF, Palmer JD, Pace NR: Perspectives on esting and worth publishing, as RPB8 was the only protein archaeal diversity, thermophily and monophyly from envi- conserved in the eukaryotic core of RNA polymerases I, II ronmental rRNA sequences. Proc Natl Acad Sci U S A 1996, 93(17):9188-9193. and III for which orthologues in archaea had not been 11. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lip- found. The fact that this protein is apparently missing man DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, from Euryarchaeota is intriguing, suggesting that either its 25(17):3389-3402. role can be fulfilled by other elements or that its primary 12. Lanzendoerfer M, Langer D, Hain J, Klenk HP, Holz I, Arnold-Ammer sequence has evolved beyond recognition. At any rate, the I, Zillig W: Structure and function of the DNA-dependent RNA polymerase of Sulfolobus . Systematic and Applied Microbiol- finding of archaeal RPB8 homologues indicates that the ogy 1994, 16:656=664. complete RNA polymerase core in both archaea and 13. She Q, Singh RK, Confalonieri F, Zivanovic Y, Allard G, Awayez MJ, eukaryotes share a common ancestry. Chan-Weiher CC, Clausen IG, Curtis BA, De Moors A, Erauso G, Fletcher C, Gordon PM, Heikamp-de Jong I, Jeffries AC, Kozera CJ, Medina N, Peng X, Thi-Ngoc HP, Redder P, Schenk ME, Theriault C, Reviewer 2: Chris Ponting, Oxford University Tolstrup N, Charlebois RL, Doolittle WF, Duguet M, Gaasterland T, Garrett RA, Ragan MA, Sensen CW, Van der Oost J: The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc This manuscript demonstrates convincingly the presence Natl Acad Sci U S A 2001, 98(14):7835-7840. of an orthologue of eukaryotic RPB8 in hyperther- 14. Andersson AF, Lundgren M, Eriksson S, Rosenlund M, Bernander R, Nilsson P: Global analysis of mRNA stability in the archaeon mophilic Crenarchaeota. As such, it provides the last miss- Sulfolobus. Genome Biol 2006, 7(10):R99. ing piece in the RNAP "puzzle" and should thus be of 15. Pei J, Kim BH, Tang M, Grishin NV: PROMALS web server for interest to many working in this field. accurate multiple protein sequence alignments. Nucleic Acids Res 2007, 35(Web Server issue):W649-52. 16. Edgar RC: MUSCLE: multiple sequence alignment with high Authors' contributions accuracy and high throughput. Nucleic Acids Res 2004, 32(5):1792-1797. EVK contributed to sequence analysis and wrote the man- 17. McGuffin LJ, Bryson K, Jones DT: The PSIPRED protein struc- uscript; KSm contributed to sequence analysis; JGE made ture prediction server. Bioinformatics 2000, 16(4):404-405. the original observation on the presence of an Rpb8

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18. Felsenstein J: Inferring phylogenies from protein sequences by parsimony, distance, and likelihood methods. Methods Enzymol 1996, 266:418-427. 19. Adachi J, Hasegawa M: MOLPHY: Programs for Molecular Phy- logenetics. Tokyo , Institute of Statistical Mathematics; 1992.

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