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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)

In addition to polynucleotide polymerization, DNA polymerases and bacterial RNA polymerase can also remove nucleotides from the growing end of nucleic acid chains. For DNA polymerases this activity is an important factor in establishing fidelity in DNA synthesis. This report describes a novel in vitro activity of RNA polymerase II whereby it cleaves an RNA chain contained within an active elongation complex. These elongation complexes are arrested at a previously identified, naturally occurring transcriptional pause site in a human gene. The new 3'-end revealed by this cleavage remains associated with an active elongation complex and is capable of being extended by RNA polymerase II. Nascent RNA cleavage is evident after removal of free nucleotides and is dependent upon a divalent metal cation and transcription elongation factor SII. This function of SII could be important in its function as an activator of transcription elongation. It is also possible that the transcript cleavage activity of RNA polymerase II represents a proofreading function of the enzyme.
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PMID:Elongation factor-dependent transcript shortening by template-engaged RNA polymerase II. 137 Dec 80

Transcription elongation in a nuclear extract in vitro is efficiently blocked by Sarkosyl at a specific site downstream of the adenovirus major late (ML) promoter at which regulated transcription arrest has also been observed in vivo. In the experiments reported here, we examined the response of the polymerase to the ML attenuation site in two assay systems: 1) purified RNA polymerase II (pol II) transcribing tailed templates and 2) elongation complexes formed on immobilized templates and then depleted of elongation factors by extensive washing. Efficient site-specific arrest occurred in both systems, demonstrating that recognition of the site is an intrinsic property of the polymerase. However, the elongation properties of washed elongation complexes and purified pol II were not equivalent. In particular, the efficiency of arrest of washed elongation complexes was influenced both by the promoter from which transcription was initiated and by DNA sequences upstream from the attenuation site that did not contribute to the arrest of purified pol II. The polymerase and washed elongation complexes both remained in stable ternary complexes at the ML site with a lifetime of hours; addition of the elongation factor SII to these complexes promoted resumption of elongation. The efficiency of arrest in both systems was dependent on the solution concentration of the nucleotide incorporated at +187 (just beyond the attenuation site), indicating that pausing is an important part of the arrest mechanism. Based on this and other findings, we argue that the polymerase assumes an altered, elongation-incompetent conformation when arrest occurs.
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PMID:Mechanistic studies of transcription arrest at the adenovirus major late attenuation site. Comparison of purified RNA polymerase II and washed elongation complexes. 137 37

The process by which RNA polymerase II elongates RNA chains remains poorly understood. Elongation factor SII is known to be required to maximize readthrough at intrinsic termination sites in vitro. We found that SII has the additional and unanticipated property of facilitating transcript cleavage by the ternary complex. We first noticed that the addition of SII caused a shortening of transcripts generated by RNA polymerase II at intrinsic termination sites during transcription reactions in which a single NTP was limiting. Truncation of the nascent transcript was subsequently observed using a series of ternary complexes artificially paused after the synthesis of 15-, 18-, 20-, 21-, and 35-nucleotide transcripts. Transcripts as short as 9 or 10 nucleotides were generated in 5-min reactions. All of these shortened RNAs remained in active ternary complexes because they could be chased quantitatively. Continuation of the truncation reaction produced RNAs as short as 4 nucleotides; however, once cleavage had proceeded to within 8 or 9 bases of the 5' end, the resulting transcription complexes could not elongate the RNAs with NTP addition. Transcript cleavage requires a divalent cation, appears to proceed primarily in 2-nucleotide increments, and is inhibited by alpha-amanitin. The catalytic site of RNA polymerase II is repositioned after transcript cleavage such that polymerization resumes at the proper location on the template strand. The extent and kinetics of the transcript truncation reaction are affected by both the position at which RNA polymerase is halted and the sequence of the transcript.
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PMID:The RNA polymerase II ternary complex cleaves the nascent transcript in a 3'----5' direction in the presence of elongation factor SII. 137 19

Regulation of transcription elongation is an important mechanism in controlling eukaryotic gene expression. SII is an RNA polymerase II-binding protein that stimulates transcription elongation and also activates nascent transcript cleavage by RNA polymerase II in elongation complexes in vitro (Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). Here we show that SII-dependent in vitro transcription through an arrest site in a human gene is preceded by nascent transcript cleavage. RNA cleavage appeared to be an obligatory step in the SII activation process. Recombinant SII activated cleavage while a truncated derivative lacking polymerase binding activity did not. Cleavage was not restricted to an elongation complex arrested at this particular site, showing that nascent RNA hydrolysis is a general property of RNA polymerase II elongation complexes. These data support a model whereby SII stimulates elongation via a ribonuclease activity of the elongation complex.
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PMID:The RNA polymerase II elongation complex. Factor-dependent transcription elongation involves nascent RNA cleavage. 137 32

DmSII is a Drosophila RNA polymerase II elongation factor which suppresses pausing by RNA polymerase II at specific sites on double stranded templates. Using antibodies produced against the purified protein, a Drosophila cDNA expression library was screened and a cDNA was isolated which encoded a portion of DmSII. When this cDNA was used to probe Kc cell mRNA the predominant species was found to be 1.4 kb in length. The original cDNA was used to screen a Drosophila Kc cell cDNA library resulting in the isolation of a 1.4 kb cDNA which was then sequenced. The deduced protein sequence for DmSII exhibited high similarity to mouse SII protein sequence. In addition, significant sequence similarity was found with the protein encoded by the yeast gene PPR2, which is involved in regulation of URA4 gene expression. The comparison of amino acid sequences suggests that DmSII is comprised of two domains homologous to mouse SII separated by a flexible, serine rich region of low homology. The shorter yeast protein has sequence similarity only to the carboxy terminal domain.
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PMID:Drosophila RNA polymerase II elongation factor DmS-II has homology to mouse S-II and sequence similarity to yeast PPR2. 224 75

Purified RNA polymerase II terminates transcription in vitro at sites within genes which also block transcript elongation in vivo. Studies on a termination site within the first intron of the human histone H3.3 gene have shown that transcription elongation factor SII can promote read-through at this site when the polymerase initiates transcription from a promoter in the presence of the accessory initiation factors. Using 3'-extended templates to direct specific initiation by purified RNA polymerase II, we show here that purified SII is sufficient to effect read-through of this terminator by the purified polymerase alone. Thus, the interaction of purified SII with an elongation complex containing only the polymerase, the template, and the nascent transcript can change the termination properties of RNA polymerase II and can effect read-through of a region that blocks elongation in the cell.
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PMID:Purified elongation factor SII is sufficient to promote read-through by purified RNA polymerase II at specific termination sites in the human histone H3.3 gene. 238 69

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.
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PMID:Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor. 239 97

Elongation and termination by RNA polymerase II are important regulatory steps for eukaryotic gene expression. We have previously studied the transcription of linear DNA templates where specific initiation of transcription by highly purified RNA polymerase II can be achieved in the absence of promoters and promoter-specific factors. Using these templates we have shown that a human histone gene, H3.3, contains sequences (intrinsic terminators) within which purified RNA polymerase II will efficiently terminate transcription (Reines, D., Wells, D., Chamberlin, M.J., and Kane, C. M. (1987) J. Mol. Biol. 196, 299-312). Curiously, these signals were found within an intron, 3'-untranslated, and protein-encoding regions of the gene suggesting that they might act to attenuate transcription of H3.3 in vivo. Here we show that intrinsic terminator sequences from an H3.3 gene intron also block in vitro transcript elongation by RNA polymerase II when the enzyme has initiated transcription from a promoter using highly purified transcription initiation factors. However, under the conditions used for promoter-specific transcription there is little transcript release. Instead the polymerase can pause at these sites for periods exceeding 60 min. We have identified and partially purified an activity from HeLa cells that causes the transcription complex to read through this block to transcription elongation. This readthrough activity fractionates with a previously characterized elongation factor (SII) over three chromatographic columns. A homogeneous preparation of calf thymus SII can also provide this activity in trans. This factor may facilitate passage of the RNA polymerase II transcription complex through such intragenic sites in cellular genes in vivo.
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PMID:Transcription elongation factor SII (TFIIS) enables RNA polymerase II to elongate through a block to transcription in a human gene in vitro. 247 7

RNA polymerase II is a multisubunit enzyme involved in the transcription of protein encoding genes. Recently acquired knowledge of the transcription process and of the RNA polymerase molecule as well as the isolation of subunit clones have led to a better understanding of the enzyme's functional regulation. Specific transcription initiation occurs at promoter regions located upstream of the gene and requires a minimum of five basic factors in addition to the enzyme. Furthermore, proteins that bind to specific DNA elements within the promoter also regulate transcriptional activity. Additional factors are required for the elongation and, possibly, termination of transcription. Two elongation factors, SII and TFIIF, interact directly with the RNA polymerase II molecule. Functional domains of RNA polymerase II have been determined by analysis of genomic clones for the two largest subunits of the enzyme. For example, the 240-kDa largest subunit contains a highly phosphorylated carboxyl-terminal heptapeptide domain repeated 26-52 times that is absolutely required for transcription in vivo. Analysis of the polymerase molecule and its interaction with basic gene-specific transcription factors will aid in our studies of the control of gene expression.
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PMID:Promoter specificity and modulation of RNA polymerase II transcription. 264 3

Genomic sequences for the large subunit of human RNA polymerase II corresponding to a part of the fifth exon were inserted into an expression vector at the carboxy-terminal end of the beta-galactosidase gene. The in-frame construct produced a 125-kilodalton fusion protein, containing approximately 10 kilodaltons of the large subunit of RNA polymerase II and 116 kilodaltons of beta-galactosidase. The purified bacterially produced fusion protein inhibited specific transcription from the adenovirus type 2 major late promoter, while beta-galactosidase had no effect. This effect of the fusion protein was during RNA elongation, not at the level of initiation, resembling the faithfully initiated but incomplete transcripts produced with purified factors in the absence of SII. Similarly, monoclonal antibody 2-7B, which reacts with the RNA polymerase II region represented in the fusion protein, inhibited specific transcription at the level of elongation in a whole-cell extract. Both monoclonal antibody 2-7B and the fusion protein, although unable to inhibit purified RNA polymerase II in a nonspecific transcription assay, selectively blocked the stimulation elicited by transcription elongation factor SII on the activity of the purified enzyme in vitro. This suggests that the fusion protein traps the SII in nonstimulatory interactions and that antibody 2-7B inhibits SII binding to RNA polymerase II. Thus, this suggests that an SII-binding contact required for specific RNA elongation resides within the fifth exon region of the largest RNA polymerase II subunit.
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PMID:Transcription elongation factor SII interacts with a domain of the large subunit of human RNA polymerase II. 314 7


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