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 polymerases I, II, and III share three subunits that are immunologically and biochemically indistinguishable. The Saccharomyces cerevisiae genes that encode these subunits (RPB5, RPB6, and RPB8) were isolated and sequenced, and their transcriptional start sites were deduced. RPB5 encodes a 25-kD protein, RPB6, an 18-kD protein, and RPB8, a 16-kD protein. These genes are single copy, reside on different chromosomes, and are essential for viability. The fact that the genes are single copy, corroborates previous evidence suggesting that each of the common subunits is identical in RNA polymerases I, II, and III. Furthermore, immunoprecipitation of RPB6 coprecipitates proteins whose sizes are consistent with RNA polymerase I, II, and III subunits. Sequence similarity between the yeast RPB5 protein and a previously characterized human RNA polymerase subunit demonstrates that the common subunits of the nuclear RNA polymerases are well conserved among eukaryotes. The presence of these conserved and essential subunits in all three nuclear RNA polymerases and the absence of recognizable sequence motifs for DNA and nucleoside triphosphate-binding indicate that the common subunits do not have a catalytic role but are important for a function shared by the RNA polymerases such as transcriptional efficiency, nuclear localization, enzyme stability, or coordinate regulation of rRNA, mRNA, and tRNA synthesis.
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PMID:Subunits shared by eukaryotic nuclear RNA polymerases. 218 66

Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10 alpha, Hs10 beta, Hs8, and Hs6, respectively), homologous to the ABC10 alpha, ABC10 beta, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10 alpha, Sc10 beta, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10 alpha and the corresponding Sp10 alpha of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-delta::HIS3) defective in Sc10 alpha. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10 beta, Sc10 beta, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGXnCCR motif presumably forms an atypical zinc-binding domain. Hs10 beta, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-delta 1::HIS3) lacking Sc10 beta. Hs8 complemented a yeast mutant (rpb8-delta 1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10 beta cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit.
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PMID:Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae. 765 87

The eukaryotic DNA-dependent RNA polymerase II (or B) is composed of 10 to 14 polypeptides ranging from 220 to 10 kDa. To gain further insight into the molecular structure and function of these subunits, we have undertaken the molecular cloning of nucleotide sequences corresponding to the human enzyme. The cDNAs of five subunits (hRPB220, hRPB140, hRPB33, hRPB25, and hRPB14.5) have been isolated. Using in situ hybridization, we show that the genes of these subunits have distinct chromosomal locations (17p13, 4q12, 16q13-q21, 19p13.3, and 19q12, respectively). Thus, if assembly of active polymerase molecules requires coordinated expression from these independent genes, mechanisms that ensure tight coregulation of the corresponding promoters must exist.
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PMID:Chromosomal localization of human RNA polymerase II subunit genes. 803 26

To assess functional relatedness of individual components of the eukaryotic transcription apparatus, three human subunits (hsRPB5, hsRPB8, and hsRPB10) were tested for their ability to support yeast cell growth in the absence of their essential yeast homologs. Two of the three subunits, hsRPB8 and hsRPB10, supported normal yeast cell growth at moderate temperatures. A fourth human subunit, hsRPB9, is a homolog of the nonessential yeast subunit RPB9. Yeast cells lacking RPB9 are unable to grow at high and low temperatures and are defective in mRNA start site selection. We tested the ability of hsRPB9 to correct the growth and start site selection defect seen in the absence of RPB9. Expression of hsRPB9 on a high-copy-number plasmid, but not a low-copy-number plasmid, restored growth at high temperatures. Recombinant human hsRPB9 was also able to completely correct the start site selection defect seen at the CYC1 promoter in vitro as effectively as the yeast RPB9 subunit. Immunoprecipitation of the cell extracts from yeast cells containing either of the human subunits that function in place of their yeast counterparts in vivo suggested that they assemble with the complete set of yeast RNA polymerase II subunits. Overall, a total of six of the seven human subunits tested previously or in this study are able to substitute for their yeast counterparts in vivo, underscoring the remarkable similarities between the transcriptional machineries of lower and higher eukaryotes.
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PMID:Six human RNA polymerase subunits functionally substitute for their yeast counterparts. 852 56

Alpha-Amanitin is a well-known specific inhibitor of RNA polymerase II (RNAPII) in vitro and in vivo. It is a cyclic octapeptide which binds with high affinity to the largest subunit of RNAPII, RPB1. We have found that in murine fibroblasts exposure to alpha-amanitin triggered degradation of the RPB1 subunit, while other RNAPII subunits, RPB5 and RPB8, remained almost unaffected. Transcriptional inhibition in alpha-amanitin-treated cells was slow and closely followed the disappearance of RPB1. The degradation rate of RPB1 was alpha-amanitin dose dependent and was not a consequence of transcriptional arrest. Alpha-Amanitin-promoted degradation of RPB1 was prevented in cells exposed to actinomycin D, another transcriptional inhibitor. Epitope-tagged recombinant human RPB1 subunits were expressed in mouse fibroblasts. In cells exposed to alpha-amanitin the wild-type recombinant subunit was degraded like the endogenous protein, but a mutated alpha-amanitin-resistant subunit remained unaffected. Hence, alpha-amanitin did not activate a proteolytic system, but instead its binding to mRPB1 likely represented a signal for degradation. Thus, in contrast to other inhibitors, such as actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, which reversibly act on transcription, inhibition by alpha-amanitin cannot be but an irreversible process because of the destruction of RNAPII.
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PMID:In vivo degradation of RNA polymerase II largest subunit triggered by alpha-amanitin. 876 Aug 75

A mutation in RPB5 (rpb5-9), an essential RNA polymerase subunit assembled into RNA polymerases I, II, and III, revealed a role for this subunit in transcriptional activation. Activation by GAL4-VP16 was impaired upon in vitro transcription with mutant whole-cell extracts. In vivo experiments using inducible reporter plasmids and Northern analysis support the in vitro data and demonstrate that RPB5 influences activation at some, but not all, promoters. Remarkably, this mutation maps to a conserved region of human RPB5 implicated by others to play a role in activation. Chimeric human-yeast RPB5 containing this conserved region now can function in place of its yeast counterpart. The defects noted with rpb5-9 are similar to those seen in truncation mutants of the RPB1-carboxyl terminal domain (CTD). We demonstrate that RPB5 and the RPB1-CTD have overlapping roles in activation because the double mutant is synthetically lethal and has exacerbated activation defects at the GAL1/10 promoter. These studies demonstrate that there are multiple activation targets in RNA polymerase II and that RPB5 and the CTD have similar roles in activation.
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PMID:RNA polymerase subunit RPB5 plays a role in transcriptional activation. 986 Sep 60

The archaeal H and eukaryotic RPB5 RNA polymerase subunits are highly homologous and are likely to play a fundamental role in transcription that extends from archaea to humans. We report the structure of subunit H, in solution, from the archaeon Methanococcus jannaschii using multidimensional nuclear magnetic resonance. The structure reveals a novel fold containing a four-stranded mixed beta sheet that is flanked on one side by three short helices. The dominant feature is beta-ribbon motif, which presents a hydrophobic, basic surface, and defines a general RNA polymerase architectural scaffold.
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PMID:RNA polymerase subunit H features a beta-ribbon motif within a novel fold that is present in archaea and eukaryotes. 1019 Nov 43

Arabidopsis thaliana contains at least four genes that are predicted to encode polypeptides related to the RPB5 subunit found in yeast and human RNA polymerase II. This subunit has been shown to be the largest subunit common to yeast RNA polymerases I, II, and III (RPABC27). More than one of these genes is expressed in Arabidopsis suspension culture cells, but only one of the encoded polypeptides is found in purified RNA polymerases II and III. This polypeptide has a predicted pI of 9.6, matches 14 of 16 amino acids in the amino terminus of cauliflower RPB5 that was microsequenced, and shows 42 and 53% amino acid sequence identity with the yeast and human RPB5 subunits, respectively.
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PMID:Arabidopsis thaliana RNA polymerase II subunits related to yeast and human RPB5. 1023 67

We previously identified a novel TATA-binding protein (TBP)-interacting protein (TIP120) from the rat liver. Here, in an RNA polymerase II (RNAP II)-reconstituted transcription system, we demonstrate that recombinant TIP120 activates the basal level of transcription from various kinds of promoters regardless of the template DNA topology and the presence of TFIIE/TFIIH and TBP-associated factors. Deletion analysis demonstrated that a 412-residue N-terminal domain, which includes an acidic region and the TBP-binding domain, is required for TIP120 function. Kinetic studies suggest that TIP120 functions during preinitiation complex (PIC) formation at the step of RNAP II/TFIIF recruitment to the promoter but not after the completion of PIC formation. Electrophoretic mobility shift assays showed that TIP120 enhanced PIC formation, and TIP120 also stimulated the nonspecific transcription and DNA-binding activity of RNAP II. These lines of evidence suggest that TIP120 is able to activate basal transcription by overcoming a kinetic impediment to RNAP II/TFIIF integration into the TBP (TFIID)-TFIIB-DNA-complex. Interestingly, TIP120 also stimulates RNAP I- and III-driven transcription and binds to RPB5, one of the common subunits of the eukaryotic RNA polymerases, in vitro. Furthermore, in mouse cells, ectopically expressed TIP120 enhances transcription from all three classes (I, II, and III) of promoters. We propose that TIP120 globally regulates transcription through interaction with basal transcription mechanisms common to all three transcription systems.
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PMID:TATA-Binding protein-interacting protein 120, TIP120, stimulates three classes of eukaryotic transcription via a unique mechanism. 1056 21

The subunits of Saccharomyces cerevisiae RNA polymerase II (RNAP II) in proximity to the DNA during transcription elongation have been identified by photoaffinity cross-linking. In the absence of transcription factors, RNAP II will transcribe a double-stranded DNA fragment containing a 3'-extension of deoxycytidines, a "tailed template". We designed a DNA template allowing the RNAP to transcribe 76 bases before it was stalled by omission of CTP in the transcription reaction. This stall site oriented the RNAP on the DNA template and allowed us to map the RNAP subunits along the DNA. The DNA analogue 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUTP (N(3)RdUTP) [Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) EMBO J. 9, 2197-205] was synthesized and enzymatically incorporated into the DNA at specified positions upstream or downstream of the stall site, in either the template or nontemplate strand of the DNA. Radioactive nucleotides were positioned beside the photoactivatable nucleotides, and cross-linking by brief ultraviolet irradiation transferred the radioactive tag from the DNA onto the RNAP subunits. In addition to N(3)RdUTP, which has a photoreactive azido group 9 A from the uridine base, we used the photoaffinity cross-linker 5N(3)dUTP with an azido group directly on the uridine ring to identify the RNAP II subunits closest to the DNA at positions where multiple subunits cross-linked. In cross-linking reactions dependent on transcription, RPB1, RPB2, and RPB5 were cross-linked with N(3)RdUTP. With 5N(3)dUTP, only RPB1 and RPB2 were cross-linked. Under certain circumstances, RPB3, RPB4, and RPB7 were cross-linked. From the information obtained in this topological study, we developed a model of yeast RNAP II in a transcription elongation complex.
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PMID:Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity cross-linking. 1106 78


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