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)

Yeast RNA polymerase II enzymes containing single amino acid substitutions in the second largest subunit were analyzed in vitro for elongation-related defects. Mutants were chosen for analysis based on their ability to render yeast cells sensitive to growth on medium containing 6-azauracil. RNA polymerase II purified from three different 6-azauracil-sensitive yeast strains displayed increased arrest at well characterized arrest sites in vitro. The extent of this defect did not correlate with sensitivity to growth in the presence of 6-azauracil. The most severe effect resulted from mutation rpb2 10 (P1018S), which occurs in region H, a domain highly conserved between prokaryotic and eukaryotic RNA polymerases that is associated with nucleotide binding. The average elongation rate of this mutant enzyme is also slower than wild type. We suggest that the slowed elongation rate and an increase in dwell time of elongating pol II leads to rpb2 10's arrest-prone phenotype. This mutant enzyme can respond to SII for transcriptional read-through and carry out SII-activated nascent RNA cleavage.
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PMID:Mutations in the second largest subunit of RNA polymerase II cause 6-azauracil sensitivity in yeast and increased transcriptional arrest in vitro. 863 12

RNA polymerases encounter specific DNA sites at which RNA chain elongation takes place in the absence of enzyme translocation in a process called discontinuous elongation. For RNA polymerase II, at least some of these sequences also provoke transcriptional arrest where renewed RNA polymerization requires elongation factor SII. Recent elongation models suggest the occupancy of a site within RNA polymerase that accommodates nascent RNA during discontinuous elongation. Here we have probed the extent of nascent RNA extruded from RNA polymerase II as it approaches, encounters, and departs an arrest site. Just upstream of an arrest site, 17-19 nucleotides of the RNA 3'-end are protected from exhaustive digestion by exogenous ribonuclease probes. As RNA is elongated to the arrest site, the enzyme does not translocate and the protected RNA becomes correspondingly larger, up to 27 nucleotides in length. After the enzyme passes the arrest site, the protected RNA is again the 18-nucleotide species typical of an elongation-competent complex. These findings identify an extended RNA product groove in arrested RNA polymerase II that is probably identical to that emptied during SII-activated RNA cleavage, a process required for the resumption of elongation. Unlike Escherichia coli RNA polymerase at a terminator, arrested RNA polymerase II does not release its RNA but can reestablish the normal elongation mode downstream of an arrest site. Discontinuous elongation probably represents a structural change that precedes, but may not be sufficient for, arrest by RNA polymerase II.
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PMID:Increased accommodation of nascent RNA in a product site on RNA polymerase II during arrest. 869 22

The toxin alpha-amanitin is frequently employed to completely block RNA synthesis by RNA polymerase II. However, we find that polymerase II ternary transcription complexes stalled by the absence of NTPs resume RNA synthesis when NTPs and amanitin are added. Chain elongation with amanitin can continue for hours at approximately 1% of the normal rate. Amanitin also greatly slows pyrophosphorolysis by elongation-competent complexes. Complexes which are arrested (that is, which have paused in transcription for long periods in the presence of excess NTPs) are essentially incapable of resuming transcription in the presence of alpha-amanitin. Complexes traversing sequences that can provoke arrest are much more likely to stop transcription in the presence of the toxin. The substitution of IMP for GMP at the 3' end of the nascent RNA greatly increases the sensitivity of stalled transcription complexes to amanitin. Neither arrested nor stalled complexes display detectable SII-mediated transcript cleavage following amanitin treatment. However, arrested complexes possess a low level, intrinsic transcript cleavage activity which is completely amanitin-resistant; furthermore, pyrophosphorolytic transcript cleavage in arrested complexes is not affected by amanitin.
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PMID:Amanitin greatly reduces the rate of transcription by RNA polymerase II ternary complexes but fails to inhibit some transcript cleavage modes. 870 41

Elongation factor SII (also known as TFIIS) is an RNA polymerase II binding protein that allows bypass of template arrest sites by activating a nascent RNA cleavage reaction. Here we show that SII contacts the 3'-end of nascent RNA within an RNA polymerase II elongation complex as detected by photoaffinity labeling. Photocross-linking was dependent upon the presence of SII, incorporation of 4-thio-UMP into RNA, and irradiation and was sensitive to treatment by RNase and proteinase. A transcriptionally active mutant of SII lacking the first 130 amino acids was also cross-linked to the nascent RNA, but SII from Saccharomyces cerevisiae, which is inactive in concert with mammalian RNA polymerase II, failed to become photoaffinity labeled. SII-RNA contact was not detected after a labeled oligoribonucleotide was released from the complex by nascent RNA cleavage, demonstrating that this interaction takes place between elongation complex-associated but not free RNA. This shows that the 3'-end of RNA is near the SII binding site on RNA polymerase II and suggests that SII may activate the intrinsic RNA hydrolysis activity by positioning the transcript in the enzyme's active site.
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PMID:Elongation factor SII contacts the 3'-end of RNA in the RNA polymerase II elongation complex. 879 87

Transcription elongation factors assist RNA polymerase II through transcriptional blockages. The human transcriptional elongation factor SII or Trascription Elongation Factor A (TCEA) releases RNA polymerase II from transcriptional arrest and is encoded by a 2.5-kb intronless gene. Using PCR primers, verified by RT-PCR to amplify the authentic, transcriptionally active SII gene, this locus was mapped to human chromosome 3 by examination of a human/rodent somatic cell hybrid panel. PCR analysis of somatic cell hybrids with chromosome 3 translocations and FISH studies utilizing a human YAC clone containing the SII gene further refine the map position of this locus to human chromosome 3p22 --> p21.3. Since another elongation factor, SIII, has been implicated in human carcinogenesis and since the interval within which the human SII gene maps is frequently deleted in certain cancers, elongation factor SII may therefore be considered a candidate gene for human malignancies involving 3p22 --> p21.3.
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PMID:Transcription elongation factor SII (TCEA) maps to human chromosome 3p22 --> p21.3. 881 34

Synthesis of eukaryotic messenger RNA by RNA polymerase II is governed by the concerted action of a set of general transcription factors that control the activity of polymerase during both the initiation and elongation stages of transcription. To date, five general elongation factors [P-TEFb, SII, TFIIF, Elongin (SIII) and ELL] have been defined biochemically. Here, we discuss these transcription factors and their roles in controlling the activity of the RNA polymerase II elongation complex.
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PMID:The RNA polymerase II general elongation factors. 887 May

RNA polymerase II contains a ribonuclease activity which is stimulated by the transcription elongation factor SII. This nuclease shortens the nascent RNA and facilitates relief of transcriptional arrest by allowing the enzyme to make multiple attempts to read through an obstacle to transcription. The catalytic center of this ribonuclease is unknown, although a region of the enzyme's second largest subunit shares local sequence similarly with barnase and other bacterial ribonucleases. To test the role of the barnase homology region in SII-activated cleavage, we engineered a single amino acid change in the Saccharomyces cerevisiae enzyme at a position homologous to a catalytic residue of barnase (Glu-371) and has been suggested as a participant in active site chemistry of RNA polymerase II. We purified RNA polymerase II from mutant yeast and assayed its ability to cleave and re-extend the nascent RNA following SII treatment. We find no defects in this function of the mutant enzyme, suggesting that the barnase homology region does not represent the active site of the SII-activated nuclease. These mutant yeast cells were also resistant to mycophenolic acid, which slows the growth of some yeast mutants bearing elongation defective RNA polymerase II or mutant elongation factor SII.
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PMID:Glutamic acid-371 of the barnase homology domain in RNA polymerase II is not required for SII-activated RNA cleavage. 903 12

We have investigated transcript elongation efficiency by RNA polymerase II on chromatin templates in vitro. Circular plasmid DNAs bearing purified RNA polymerase II transcription complexes were assembled into nucleosomes using purified histones and transient exposure to high salt, followed by dilution and dialysis. This approach resulted in nucleosome assembly beginning immediately downstream of the transcription complexes. RNA polymerases on these nucleosomal templates could extend their 15- or 35-nucleotide nascent RNAs by only about 10 nucleotides in 15 min, even in the presence of elongation factors TFIIF and SII. Efficient transcript elongation did occur upon dissociation of nucleosomes with 1% sarkosyl, indicating that the RNA polymerases were not damaged by the high salt reconstitution procedure. Since the elongation complexes were released by sarkosyl but not by SII, these complexes apparently did not enter the arrested conformation when they encountered nucleosomes. Surprisingly, elongation was no more efficient on nucleosomal templates reconstituted only with H3/H4 tetramers, even in the presence of elongation factors and/or competitor DNA at high concentration. Thus, in a purified system lacking nucleosome remodeling factors, not only the core histone octamer but also the H3/H4 tetramer provide an nearly absolute block to transcript elongation by RNA polymerase II, even in the presence of elongation factors.
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PMID:The H3/H4 tetramer blocks transcript elongation by RNA polymerase II in vitro. 928 58

An important control point for gene regulation is the frequency of initiations leading to different numbers of RNA polymerases simultaneously transcribing the same gene. To date, the only direct assay for multiple-round transcription by RNA polymerase II in vitro required G-free cassette-containing templates and GTP-free conditions and was thus restricted in application. Here we used instead templates containing a triplex-directed interstrand psoralen-DNA cross-link to block RNA polymerase II elongation at a specific location. Covalently cross-linked templates allowed simultaneous detection of both specific initiation and reinitiation with any combination of promoter and transcribed sequence. In reconstituted systems, identical stacking of RNA polymerases was observed when the first polymerase was halted by GTP deprivation at the end of a G-free cassette or by a covalent cross-link downstream of different transcribed sequences. In contrast to transcription of G-free cassettes, reinitiation was unaffected by the transcription factor SII on sequences containing all four nucleotides. In crude nuclear extracts, transcription of covalently cross-linked templates yielded a reinitiation pattern with a wider spacing than in more purified fractions, indicating that the elongation complexes from nuclear extract contained a different form of RNA polymerase II or a different complement of associated factors.
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PMID:Multiple rounds of transcription by RNA polymerase II at covalently cross-linked templates. 959 65

We have addressed whether the intrinsic 3'-->5' nuclease activity of human RNA polymerase II (pol II) can proofread during transcription in vitro. In the presence of SII, a protein that stimulates the nuclease activity, pol II quantitatively removed misincorporated nucleotides from the nascent transcript during rapid chain extension. The basis of discrimination between the correct and incorrect base was the slow addition of the next nucleotide to the mismatched terminus. Incorporation of inosine monophosphate inhibited next nucleotide addition by a similar magnitude as a mismatched base. We used this finding to demonstrate that addition of SII to a transcription reaction dramatically altered the RNA base content, reflecting the stable incorporation of more "correct" (GMP) and fewer "incorrect" (IMP) nucleotides.
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PMID:Transcriptional fidelity and proofreading by RNA polymerase II. 960 37


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