NORMAL GENOMIC

• Grohmann D, Werner F
• Cycling through transcription with the RNA polymerase F/E (RPB4/7)complex: structure, function and evolution of archaeal RNA polymerase.
• Res Microbiol. 2011; 162: 10-8
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• RNA polymerases (RNAPs) from the three domains of life, Bacteria, Archaeaand Eukarya, are evolutionarily related and thus have common structuraland functional features. Despite the radically different morphology ofArchaea and Eukarya, their RNAP subunit composition and utilisation ofbasal transcription factors are almost identical. This review focuses onthe multiple functions of the most prominent feature that differentiatesthese enzymes from the bacterial RNAP--a stalk-like protrusion, whichconsists of the heterodimeric F/E subcomplex. F/E is highly versatile, itfacilitates DNA strand-separation during transcription initiation,increases processivity during the elongation phase of transcription andensures efficient transcription termination.

• Goto-Ito S, Ito T, Ishii R, Muto Y, Bessho Y, Yokoyama S
• Crystal structure of archaeal tRNA(m(1)G37)methyltransferase aTrm5.
• Proteins. 2008; 72: 1274-89
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• Methylation of the N1 atom of guanosine at position 37 in tRNA, theposition 3'-adjacent to the anticodon, generates the modified nucleosidem(1)G37. In archaea and eukaryotes, m(1)G37 synthesis is catalyzed bytRNA(m(1)G37)methyltransferase (archaeal or eukaryotic Trm5, a/eTrm5).Here we report the crystal structure of archaeal Trm5 (aTrm5) fromMethanocaldococcus jannaschii (formerly known as Methanococcus jannaschii)in complex with the methyl donor analogue at 2.2 A resolution. The crystalstructure revealed that the entire protein is composed of three structuraldomains, D1, D2, and D3. In the a/eTrm5 primary structures, D2 and D3 arehighly conserved, while D1 is not conserved. The D3 structure is theRossmann fold, which is the hallmark of the canonical class-Imethyltransferases. The a/eTrm5-defining domain, D2, exhibits structuralsimilarity to some class-I methyltransferases. In contrast, a DALI searchwith the D1 structure yielded no structural homologues. In the crystalstructure, D3 contacts both D1 and D2. The residues involved in the D1:D3interactions are not conserved, while those participating in the D2:D3interactions are well conserved. D1 and D2 do not contact each other, andthe linker between them is disordered. aTrm5 fragments corresponding tothe D1 and D2-D3 regions were prepared in a soluble form. The NMR analysisof the D1 fragment revealed that D1 is well folded by itself, and it didnot interact with either the D2-D3 fragment or the tRNA. The NMR analysisof the D2-D3 fragment revealed that it is well folded, independently ofD1, and that it interacts with tRNA. Furthermore, the D2-D3 fragment wasas active as the full-length enzyme for tRNA methylation. The positivecharges on the surface of D2-D3 may be involved in tRNA binding.Therefore, these findings suggest that the interaction between D1 and D3is not persistent, and that the D2-D3 region plays the major role in tRNAmethylation.

• Knutson BA, Broyles SS
• Expansion of poxvirus RNA polymerase subunits sharing homology withcorresponding subunits of RNA polymerase II.
• Virus Genes. 2008; 36: 307-11
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• Poxvirus-encoded RNA polymerases were known previously to share extensivesequence homology in their two largest subunits with the correspondingsubunits of cellular RNA polymerases and a modest alignment between thesmallest poxvirus subunit and RBP10 of RNA polymerase II. The remainingsubunits had no apparent cellular homologs. In this study, the HHpredprogram that combines amino acid sequence alignments with secondarystructure predictions was used to search for homologs to the poxvirus RNApolymerase subunits. Significant matches of vaccinia RNA polymerase 22-,19-, and 18-kDa subunits to RNA polymerase II subunits RPB5, 6, and 7,respectively, were identified. These results strengthen the concept thatpoxviral RNA polymerases likely evolved from cellular RNA polymerases.

• Boomershine WP, McElroy CA, Tsai HY, Wilson RC, Gopalan V, Foster MP
• Structure of Mth11/Mth Rpp29, an essential protein subunit of archaeal andeukaryotic RNase P.
• Proc Natl Acad Sci U S A. 2003; 100: 15398-403
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• We have determined the solution structure of Mth11 (Mth Rpp29), anessential subunit of the RNase P enzyme from the archaebacteriumMethanothermobacter thermoautotrophicus (Mth). RNase P is a ubiquitousribonucleoprotein enzyme primarily responsible for cleaving the 5' leadersequence during maturation of tRNAs in all three domains of life. Ineubacteria, this enzyme is made up of two subunits: a large RNA (approximately 120 kDa) responsible for mediating catalysis, and a smallprotein cofactor ( approximately 15 kDa) that modulates substraterecognition and is required for efficient in vivo catalysis. In contrast,multiple proteins are associated with eukaryotic and archaeal RNase P, andthese proteins exhibit no recognizable homology to the conserved bacterialprotein subunit. In reconstitution experiments with recombinantlyexpressed and purified protein subunits, we found that Mth Rpp29, ahomolog of the Rpp29 protein subunit from eukaryotic RNase P, is anessential protein component of the archaeal holoenzyme. Consistent withits role in mediating protein-RNA interactions, we report that Mth Rpp29is a member of the oligonucleotide/oligosaccharide binding fold family. Inaddition to a structured beta-barrel core, it possesses unstructured N-and C-terminal extensions bearing several highly conserved amino acidresidues. To identify possible RNA contacts in the protein-RNA complex, weexamined the interaction of the 11-kDa protein with the full 100-kDa MthRNA subunit by using NMR chemical shift perturbation. Our findingsrepresent a critical step toward a structural model of the RNase Pholoenzyme from archaebacteria and higher organisms.

• Mura C, Cascio D, Sawaya MR, Eisenberg DS
• The crystal structure of a heptameric archaeal Sm protein: Implicationsfor the eukaryotic snRNP core.
• Proc Natl Acad Sci U S A. 2001; 98: 5532-7
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• Sm proteins form the core of small nuclear ribonucleoprotein particles(snRNPs), making them key components of several mRNA-processingassemblies, including the spliceosome. We report the 1.75-A crystalstructure of SmAP, an Sm-like archaeal protein that forms a heptamericring perforated by a cationic pore. In addition to providing directevidence for such an assembly in eukaryotic snRNPs, this structure (i)shows that SmAP homodimers are structurally similar to human Smheterodimers, (ii) supports a gene duplication model of Sm proteinevolution, and (iii) offers a model of SmAP bound to single-stranded RNA(ssRNA) that explains Sm binding-site specificity. The pronouncedelectrostatic asymmetry of the SmAP surface imparts directionality toputative SmAP-RNA interactions.

• Lai L, Yokota H, Hung LW, Kim R, Kim SH
• Crystal structure of archaeal RNase HII: a homologue of human major RNaseH.
• Structure. 2000; 8: 897-904
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• BACKGROUND: RNases H are present in all organisms and cleave RNAs inRNA/DNA hybrids. There are two major types of RNases H that have littlesimilarity in sequence, size and specificity. The structure of RNase HI,the smaller enzyme and most abundant in bacteria, has been extensivelystudied. However, no structural information is available for the largerRNase H, which is most abundant in eukaryotes and archaea. Mammalian RNaseH participates in DNA replication, removal of the Okazaki fragments andpossibly DNA repair. RESULTS: The crystal structure of RNase HII from thehypothermophile Methanococcus jannaschii, which is homologous to mammalianRNase H, was solved using a multiwavelength anomalous dispersion (MAD)phasing method at 2 A resolution. The structure contains two compactdomains. Despite the absence of sequence similarity, the large N-terminaldomain shares a similar fold with the RNase HI of bacteria. The activesite of RNase HII contains three aspartates: Asp7, Asp112 and Asp149. Thenucleotide-binding site is located in the cleft between the N-terminal andC-terminal domains. CONCLUSIONS: Despite a lack of any detectablesimilarity in primary structure, RNase HII shares a similar structuraldomain with RNase HI, suggesting that the two classes of RNases H have acommon catalytic mechanism and possibly a common evolutionary origin. Theinvolvement of the unique C-terminal domain in substrate recognitionexplains the different reaction specificity observed between the twoclasses of RNase H.

• Sheffer A, Varon M, Choder M
• Rpb7 can interact with RNA polymerase II and support transcription duringsome stresses independently of Rpb4.
• Mol Cell Biol. 1999; 19: 2672-80
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• Rpb4 and Rpb7 are two yeast RNA polymerase II (Pol II) subunits whosemechanistic roles have recently started to be deciphered. Althoughprevious data suggest that Rpb7 can stably interact with Pol II only as aheterodimer with Rpb4, RPB7 is essential for viability, whereas RPB4 isessential only during some stress conditions. To resolve this discrepancyand to gain a better understanding of the mode of action of Rpb4, we tookadvantage of the inability of cells lacking RPB4 (rpb4Delta, containingPol IIDelta4) to grow above 30 degrees C and screened for genes whoseoverexpression could suppress this defect. We thus discovered thatoverexpression of RPB7 could suppress the inability of rpb4Delta cells togrow at 34 degrees C (a relatively mild temperature stress) but not athigher temperatures. Overexpression of RPB7 could also partially suppressthe cold sensitivity of rpb4Delta strains and fully suppress theirinability to survive a long starvation period (stationary phase). Notably,however, overexpression of RPB4 could not override the requirement forRPB7. Consistent with the growth phenotype, overexpression of RPB7 couldsuppress the transcriptional defect characteristic of rpb4Delta cellsduring the mild, but not during a more severe, heat shock. We alsodemonstrated, through two reciprocal coimmunoprecipitation experiments, astable interaction of the overproduced Rpb7 with Pol IIDelta4.Nevertheless, fewer Rpb7 molecules interacted with Pol IIDelta4 than withwild-type Pol II. Thus, a major role of Rpb4 is to augment the interactionof Rpb7 with Pol II. We suggest that Pol IIDelta4 contains a small amountof Rpb7 that is sufficient to support transcription only under nonstressconditions. When RPB7 is overexpressed, more Rpb7 assembles with PolIIDelta4, enough to permit appropriate transcription also under somestress conditions.

• Maillet I, Buhler JM, Sentenac A, Labarre J
• Rpb4p is necessary for RNA polymerase II activity at high temperature.
• J Biol Chem. 1999; 274: 22586-90
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• Rpb4p and Rpb7p are two subunits of the yeast RNA polymerase II, whichform a subcomplex that can dissociate from the enzyme in vitro. WhereasRPB7 is essential, RPB4 is dispensable for cellular viability. However,the rpb4 null mutant is heat-sensitive, and it has been suggested thatRpb4p is an essential component for cellular stress response. To examinethis hypothesis, we used two-dimensional gel electrophoresis to analyzethe protein expression pattern of the rpb4 null mutant in response to heatshock, oxidative stress, osmotic stress, and in the post-diauxic phase. Weshow that this mutant is not impaired in stress induced transcriptionalactivation: the absence of heat shock response of the mutant is due to ageneral defect in RNA polymerase II activity at high temperature. Underthis condition, Rpb4p is necessary to maintain the polymerase activity invivo. The heat growth defect of the rpb4 null mutant can be partiallysuppressed by overexpression of RPB7, suggesting that Rpb4p maintains orstabilizes Rpb7p in the RNA polymerase. We also demonstrate that rpb4 nullmutant is an appropriate tool to analyze the involvement oftranscriptional events in the survival and adaptation to heat shock orother stresses.

• Bateman A
• The structure of a domain common to archaebacteria and the homocystinuriadisease protein.
• Trends Biochem Sci. 1997; 22: 12-3