Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNA polymerase II from the fission yeast Schizo-saccharomyces pombe consists of 12 species of subunits, Rpb1-Rpb12. We expressed these subunits, except Rpb4, simultaneously in cultured insect cells with baculovirus expression vectors. For the isolation of subunit complexes formed in the virus-infected cells, a glutathione S -transferase (GST) sequence was fused to the rpb3 cDNA to produce GST-Rpb3 fusion protein and a decahistidine-tag sequence was inserted into the rpb1 cDNA to produce Rpb1H protein. After successive affinity chromatography on glutathione and Ni(2+)columns, complexes consisting of the seven subunits, Rpb1H, Rpb2, GST-Rpb3, Rpb5, Rpb7, Rpb8 and Rpb11, were identified. Omission of the GST-Rpb3 expression resulted in reduced assembly of the Rpb11 into the complex. Direct interaction between Rpb3 and the other six subunits was detected by pairwise coexpression experiments. Coexpression of various combinations of a few subunits revealed that Rpb11 enhances Rpb3-Rpb8 interaction and consequently Rpb8 enhances Rpb1-Rpb3 interaction to some extent. We propose a mechanism in which the assembly of RNA poly-merase II is stabilized through multiple subunit-subunit contacts.
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PMID:Involvement of multiple subunit-subunit contacts in the assembly of RNA polymerase II. 1064 88

Protein splicing is a self-catalytic process in which an intervening sequence, termed an intein, is excised from a protein precursor, and the flanking polypeptides are religated. The conserved intein penultimate His facilitates this reaction by assisting in Asn cyclization, which results in C-terminal splice junction cleavage. However, many inteins do not have a penultimate His. Previous splicing studies with 2 such inteins yielded contradictory results. To resolve this issue, the splicing capacity of 2 more inteins without penultimate His residues was examined. Both the Methanococcus jannaschii phosphoenolpyruvate synthase and RNA polymerase subunit A' inteins spliced. Splicing of the phosphoenolpyruvate synthase intein improved when its penultimate Phe was changed to His, but splicing of the RNA polymerase subunit A' intein was inhibited when its penultimate Gly was changed to His. We propose that inteins lacking a penultimate His (i) arose by mutation from ancestors in which a penultimate His facilitated splicing, (ii) that loss of this His inhibited, but may not have blocked, splicing, and (iii) that selective pressure for efficient expression of the RNA polymerase yielded an intein that utilizes another residue to assist Asn cyclization, changing the intein active site so that a penultimate His now inhibits splicing.
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PMID:Protein splicing in the absence of an intein penultimate histidine. 1077 Sep 23

Protein interactions among RNA polymerase small subunits from the archaeon Methanococcus jannaschii were investigated using affinity pulldown assays in pairwise and higher-order combinations. In the most extensive study of archaeal RNA polymerase subunit interactions to date, including 37 pairs of proteins, 10 ternary combinations, and three quaternary combinations, we found evidence for pairwise interactions of subunit D with subunits L and N, and a ternary complex of subunits D, L and N. No other small subunit interactions occurred. These results are consistent with interactions observed in a crystal structure of eukaryotic RNA polymerase II and support a common archaeal/eukaryal RNA polymerase architecture. We further propose that subunit E" is not an integral member of archaeal RNA polymerases. Finally, we discuss the relative accuracy of the various methods that have been used to predict protein-protein interactions in RNA polymerase.
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PMID:Similar subunit architecture of archaeal and eukaryal RNA polymerases. 1116

The RNA polymerase II (Pol II) of eukaryotes is composed of 12 subunits, of which five are shared among Pol I, Pol II and Pol III. At present, however, little is known about the regulation of synthesis and assembly of the 12 Pol II subunits. To obtain an insight into the regulation of synthesis of these 12 Pol II subunits, Rpb1 to Rpb12, in the fission yeast Schizosaccharomyces pombe, we analysed the transcriptional organization of the rpb genes by use of the oligo capping method, and determined mRNA levels by quantitative competitive PCR assay. The intracellular concentrations of the 12 Rpb subunits in growing S. pombe cells are different, within a range of 15-fold difference between the least abundant Rpb3 and the most abundant Rpb12. The transcription of one group of genes including rpb3, rpb4, rpb5, rpb6, rpb7 and rpb10 is mainly initiated at a single site, while that of the other group of genes for rpb1, rpb2, rpb8, rpb9, rpb11 and rpb12 is initiated at multiple sites. The promoters of the first group of genes contain the TATA box sequence between -26 and -62, while the second group of genes carry TATA-less promoters. Several common sequence segments, tentatively designated 'Rpb motifs', were identified in the promoter regions of the rpb genes. Competitive PCR analysis indicated that mRNAs for Rpb1, Rpb3, Rpb7 and Rpb9 were among the group which had a low abundance, while the levels of Rpb6 and Rpb10 mRNAs were about fivefold, and that of Rpb2 mRNA was about 40-fold higher than the Rpb3 mRNA level. The levels of rpb mRNAs do not correlate with those of Rpb proteins. The protein-to-mRNA ratio or the translation efficiency is low for the rpb1, rpb2, rpb3 and rpb11 genes, encoding the homologues of subunits beta', beta, alpha and alpha, respectively, of the prokaryotic RNA polymerase core enzyme.
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PMID:Transcription organization and mRNA levels of the genes for all 12 subunits of the fission yeast RNA polymerase II. 1116 94

Ralstonia metallidurans CH34 can use biphenyl as carbon and energy source when provided with the catabolic transposon Tn4371. Previous results suggested that this property was dependent on the RNA polymerase subunit sigma(54). The authors sequenced the CH34 rpoN gene and flanking DNA and isolated a CH34 rpoN-deficient strain. Analysis of the sequence revealed a set of features conserved in all rpoN genes and flanking DNA regions previously analysed in other bacterial species. Nevertheless, despite this conservation, CH34 differed even from the closely related strain R. eutropha H16 by one particular ORF. The rpoN null mutation did not affect expression of the Tn4371 bph operon although it did alter the ability of the Tn4371 host strain to grow on biphenyl. The CH34 rpoN mutant had lost the capacity for autotrophic growth and for responding to poor nitrogen sources by a decrease in urease and proline oxidase activity. CH34 RNA polymerase sigma(54) thus positively controls autotrophy as well as nitrogen metabolism but only indirectly affects Tn4371-directed biphenyl utilization.
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PMID:Ralstonia metallidurans CH34 RpoN sigma factor and the control of nitrogen metabolism and biphenyl utilization. 1142 71

Studies of the Escherichia coli RNA polymerase subunit sigma-70 employing limited proteolytic digestion and binding by monoclonal antibodies indicate that conserved region 3 is solvent accessible in the free protein and in the RNA polymerase holoenzyme. Conversely, when sigma-70 binds to core RNA polymerase, proteolytic cleavage of region 3 is dramatically reduced. The former set of results seems to indicate the physical presence of region 3 on or near the surface of the holoenzyme while the latter of these results suggest that region 3 is sequestered in a direct protein-protein contact within the RNA holoenzyme which alters its protease sensitivity. To further investigate these possibilities we inserted an internal histidine-tag within region 3 of sigma(70) (sigma(70)-R3-His6) between amino acids 508 and 509. Confirmation that the internal His-tag insertion does not disrupt normal sigma(70) function was verified by genetic complementation. His-tagged protein was immobilized on nickel-agarose and core RNAP was tethered via the sigma-core interaction. Our results are consistent with the localization of region 3 on or near the surface both of free sigma(70) and of RNA polymerase holoenzyme. Furthermore, we find that the sigma(70)-core interaction is resistant to high ionic conditions but is completely disrupted by the presence of the low-molecular-weight hydrophobic amino acids phenylalanine and leucine free in solution. These results demonstrate the general usefulness of this approach to the disruption of protein-protein interactions and its potential application for protein purification.
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PMID:Low concentrations of free hydrophobic amino acids disrupt the Escherichia coli RNA polymerase core-sigma(70) protein-protein interaction. 1181 37

In transcriptional regulation, RNA polymerase II (pol II) interacts and forms complexes with a number of protein factors. To isolate and identify the pol II-associated proteins, we constructed a Schizosaccharomyces pombe strain carrying a FLAG tag sequence fused to the rpb3 gene encoding the pol II subunit Rpb3. By immunoaffinity purification with anti-FLAG antibody-resin, a pol II complex containing the Rpb1 subunit with a nonphosphorylated carboxyl-terminal domain (CTD) was isolated. In addition to the pol II subunits, the complex was found to contain three subunits of a transcription factor TFIIF (TFIIF alpha, TFIIF beta, and Tfg3) and TFIIF-interacting CTD-phosphatase Fcp1. The same type of pol II complex could also be purified from an Fcp1-tagged strain. The isolated Fcp1 showed CTD-phosphatase activity in vitro. The fcp1 gene is essential for cell viability. Fcp1 and pol II interacted directly in vitro. Furthermore, by chemical cross-linking, glutathione S-transferase pulldown, and affinity chromatography, the Fcp1-interacting subunit of pol II was identified as Rpb4, which plays regulatory roles in transcription. We also constructed an S. pombe thiamine-dependent rpb4 shut-off system. On repression of rpb4 expression, the cell produced more of the nonphosphorylated form of Rpb1, but the pol II complex isolated with the anti-FLAG antibody contained less Fcp1 and more of the phosphorylated form of Rpb1 with a concomitant reduction in Rpb4. This result indicates the importance of Fcp1-Rpb4 interaction for formation of the Fcp1/TFIIF/pol II complex in vivo.
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PMID:Formation of a carboxy-terminal domain phosphatase (Fcp1)/TFIIF/RNA polymerase II (pol II) complex in Schizosaccharomyces pombe involves direct interaction between Fcp1 and the Rpb4 subunit of pol II. 1183 23

Using structural comparisons, we identified a novel domain with a simple fold in the bacterial cell division ATPase FtsA, the archaeo-eukaryotic RNA polymerase subunit Rpb7p, the GyrI superfamily, and the uncharacterized MTH1598/Tm1083-like proteins. The fold contains a core of 3 strands, forming a curved sheet, and a single helix in a strand-helix-strand-strand (SHS2) configuration. The SHS2 domain may exist either in single or duplicate copies within the same polypeptide. The single-copy versions of the domain in FtsA and Rbp7p are most closely related, and appear to mediate protein-protein interactions by means of strand 1, and the loop between strand 2 and strand 3 of the domain. We predict that the interactions between FtsA and its functional partners in bacterial cell division are likely to be similar to the interactions of Rbp7p in the archaeo-eukaryotic RNA polymerase complex. The dimeric versions typified by the GyrI superfamily appear to have been adapted for small-molecule binding. Sequence profiles searches helped us to identify several new versions of the GyrI superfamily, including a family of secreted forms that is found only in animals and the bacterial pathogen Leptospira. Through sequence-structure comparisons, we predict the positions that are likely to be important for ligand specificity in the GyrI superfamily. In the MTH1598/Tm1083-like proteins, a SHS2 domain is inserted into the loop between strand 1 and helix 1 of another SHS2 domain. This has resulted in a structure that has convergent similarities with the Hsp33 and green fluorescent protein folds. The sequence conservation pattern and its phyletic profile suggest that it might function as an enzyme in some conserved aspect of nucleic acid metabolism. Thus, the SHS2 domain is an example of a simple module that has been adapted to perform an entire spectrum of functions ranging from protein-protein interactions to small-molecule recognition and catalysis.
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PMID:The SHS2 module is a common structural theme in functionally diverse protein groups, like Rpb7p, FtsA, GyrI, and MTH1598/TM1083 superfamilies. 1528 Nov 31

Emiliania huxleyi virus strain 86 is the largest algal virus sequenced to date and is unique among the Phycodnaviridae since its genome is predicted to contain six RNA polymerase subunit genes. We have used a virus microarray to profile the temporal transcription strategy of this unusual virus during infection. There are two distinct transcription phases to the infection process. The primary phase is dominated by a group of coding sequences (CDSs) expressed by 1 h postinfection that are localized to a subregion of the genome. The CDS of the primary group have no database homologues, and each is associated with a unique promoter element. The remainder of the CDSs are expressed in a secondary phase between 2 and 4 hours postinfection. Compartmentalized transcription of the two distinctive phases is discussed. We hypothesize that immediately after infection the nucleic acid of the virus targets the host nucleus, where primary-phase genes are transcribed by host RNA polymerase which recognizes the viral promoter. Secondary-phase transcription may then be conducted in the cytoplasm.
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PMID:Locus-specific gene expression pattern suggests a unique propagation strategy for a giant algal virus. 1684 Mar 48

Treatment of wild-type vaccinia virus infected cells with the anti-poxviral drug isatin-beta-thiosemicarbazone (IBT) induces the viral postreplicative transcription apparatus to synthesize longer-than-normal mRNAs through an unknown mechanism. Prior studies have shown that virus mutants resistant to or dependent on IBT affect proteins involved in control of viral postreplicative transcription elongation, including G2, J3, and the viral RNA polymerase. Prior studies also suggest that there exist additional unidentified vaccinia genes that influence transcription elongation. The present study was undertaken to target candidate transcription elongation factor genes in an error-prone mutagenesis protocol to determine whether IBT-resistant or -dependent alleles could be isolated in those candidate genes. Mutagenesis of genes in which IBT resistance alleles have previously been isolated, namely A24R (encoding the second largest RNA polymerase subunit, rpo132) and G2R (encoding a positive transcription elongation factor), resulted in isolation of novel IBT resistance and dependence alleles therefore providing proof of principle of the targeted mutagenesis technique. The vaccinia H5 protein has been implicated previously in transcription elongation by virtue of its association with the positive elongation factor G2. Mutagenesis of the vaccinia H5R gene resulted in a novel H5R IBT resistance allele, strongly suggesting that H5 is a positive transcription elongation factor.
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PMID:A targeted approach to identification of vaccinia virus postreplicative transcription elongation factors: genetic evidence for a role of the H5R gene in vaccinia transcription. 1737 1


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