Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA Polymerase II”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources
PubMed1992

A novel transcription factor reveals a functional link between the RNA polymerase II CTD and TFIID.

The RNA polymerase II large subunit carboxy-terminal domain (CTD) plays a role in transcription initiation, but its mechanism of action is not well understood. We have investigated the function of the SRB2 gene, which was isolated as a dominant suppressor of CTD truncation mutations. The allele specificity of this suppressor indicates that SRB2 and the CTD are involved in the same function. Indeed, cells lacking SRB2 and cells lacking a large portion of the CTD exhibit the same set of conditional growth phenotypes and exhibit very similar defects in gene expression in vivo. The SRB2 protein is a novel transcription factor that has an important role in basal and activated transcription in vitro and is essential for efficient establishment of the transcription initiation apparatus. Template commitment experiments suggest that SRB2 becomes physically associated with the transcription initiation complex. We find that SRB2 binds specifically to TFIID. As SRB2 and the RNA polymerase II CTD are involved in the same function, these results reveal a functional link between the CTD and the TATA-binding factor. This study implicates the CTD in recruitment of RNA polymerase II to the transcription initiation complex.

Amino Acid Sequence↗
PubMed2005

The highly conserved glutamic acid 791 of Rpb2 is involved in the binding of NTP and Mg(B) in the active center of human RNA polymerase II.

During transcription by RNA polymerase (RNAP) II, the incoming ribonucleoside triphosphate (NTP) enters the catalytic center in association with an Mg2+ ion, termed metal B [Mg(B)]. When bound to RNAP II, Mg(B) is coordinated by the beta and gamma phosphates of the NTP, Rpb1 residues D481 and D483 and Rpb2 residue D837. Rpb2 residue D837 is highly conserved across species. Notably, its neighboring residue, E836 (E791 in human RNAP II), is also highly conserved. To probe the role of E791 in transcription, we have affinity purified and characterized a human RNAP II mutant in which this residue was substituted for alanine. Our results indicate that the transcription activity of the Rpb2 E791A mutant is impaired at low NTP concentrations both in vitro and in vivo. They also revealed that both its NTP polymerization and transcript cleavage activities are decreased at low Mg concentrations. Because Rpb2 residue E791 appears to be located too far from the NTP-Mg(B) complex to make direct contact at either the entry (E) or addition (A) site, we propose alternative mechanisms by which this highly conserved residue participates in loading NTP-Mg(B) in the active site during transcription.

Amino Acid Substitution↗
PubMed1994

Inhibitors of transcription such as 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole and isoquinoline sulfonamide derivatives (H-8 and H-7) promote dephosphorylation of the carboxyl-terminal domain of RNA polymerase II largest subunit.

The RNA polymerase IIO and IIA differ by the extent of phosphorylation in the carboxyl-terminal domain (CTD) of the largest subunit. It has been proposed that the IIA form of RNA polymerase II interacts with the promoter to form a stable preinitiation complex whereas the IIO form would be generated upon entry into initiation of transcription. Phosphorylation of the CTD might be required to release the interaction between the polymerase and the promoter binding factors. In this paper, we show that in the presence of actinomycin D, the phosphorylated IIO form accumulates. In contrast, the dephosphorylated IIA form accumulates while the amount of phosphorylated IIo form decreases in cells treated with CTD-kinase inhibitors such as the nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole or the isoquinoline sulfonamide derivatives H-7* or H-8. These changes are fast and suggest a very rapid phosphate turnover on the CTD. Transcription is inhibited in intact cells by drug concentrations that are effective in altering CTD phosphorylation, although no causal relationship is established yet. These effects do not concern other cellular functions such as protein synthesis. Thus isoquinoline sulfonamide derivatives might be helpful to further dissect the role of CTD phosphorylation in transcription.

Dactinomycin↗
PubMed2002

The transcription cycle of RNA polymerase II in living cells.

RNA polymerase II transcribes most eukaryotic genes. Its catalytic subunit was tagged with green fluorescent protein and expressed in Chinese hamster cells bearing a mutation in the same subunit; it complemented the defect and so was functional. Photobleaching revealed two kinetic fractions of polymerase in living nuclei: approximately 75% moved rapidly, but approximately 25% was transiently immobile (association t1/2 approximately 20 min) and transcriptionally active, as incubation with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole eliminated it. No immobile but inactive fraction was detected, providing little support for the existence of a stable holoenzyme, or the slow stepwise assembly of a preinitiation complex on promoters or the nuclear substructure. Actinomycin D decreased the rapidly moving fraction, suggesting that engaged polymerases stall at intercalated molecules while others initiate. When wild-type cells containing only the endogenous enzyme were incubated with [3H]uridine, nascent transcripts became saturated with tritium with similar kinetics (t1/2 approximately 14 min). These data are consistent with a polymerase being mobile for one half to five sixths of a transcription cycle, and rapid assembly into the preinitiation complex. Then, most expressed transcription units would spend significant times unassociated with engaged polymerases.

Animals↗
PubMed2005

Distinct regions of RPB11 are required for heterodimerization with RPB3 in human and yeast RNA polymerase II.

In Saccharomyces cerevisiae, RNA polymerase II assembly is probably initiated by the formation of the RPB3-RPB11 heterodimer. RPB3 is encoded by a single copy gene in the yeast, mouse and human genomes. The RPB11 gene is also unique in yeast and mouse, but in humans a gene family has been identified that potentially encodes several RPB11 proteins differing mainly in their C-terminal regions. We compared the abilities of both yeast and human proteins to heterodimerize. We show that the yeast RPB3/RPB11 heterodimer critically depends on the presence of the C-terminal region of RPB11. In contrast, the human heterodimer tolerates significant changes in RPB11 C-terminus, allowing two human RPB11 variants to heterodimerize with the same efficiency with RPB3. In keeping with this observation, the interactions between the conserved N-terminal 'alpha-motifs' is much more important for heterodimerization of the human subunits than for those in yeast. These data indicate that the heterodimerization interfaces have been modified during the course of evolution to allow a recent diversification of the human RPB11 subunits that remains compatible with heterodimerization with RPB3.

Amino Acid Motifs↗
PubMed2006

Exon tethering in transcription by RNA polymerase II.

There is an emerging consensus that RNA polymerase II (RNA Pol II) transcription and pre-mRNA processing are tightly coupled events. We show here that exons flanking an intron that has been engineered to be co-transcriptionally cleaved are accurately and efficiently spliced together. These data underline the close coupling of processes in the initial stages of protein-encoding gene expression and provide evidence for a molecular tether connecting emergent splice sites in the pre-mRNA to transcribing RNA Pol II. This observation suggests that for some genes a continuous intron transcript is not required for pre-mRNA splicing in vivo.

Animals↗
PubMed1986

RNA contacts subunits IIo and IIc in HeLa RNA polymerase II transcription complexes.

Subunits of RNA polymerase II that come into contact with nascent RNA transcripts have been determined by photoaffinity labeling. Transcription was carried out in a cell-free extract in the presence of 4-thio-UTP, and utilized the major late promoter of adenovirus-2 DNA. The transcript length was limited by inclusion of the chain terminator 3'-O-methyl-GTP. Transcription complexes were irradiated with near-UV light (lambda greater than 300 nm) to specifically photoactivate 4-thio-uridine. After photocross-linking, radiolabeled proteins were separated by electrophoresis, blotted onto nitrocellulose, and visualized by autoradiography. Specific photoaffinity labeling of enzyme subunits IIo and IIc was observed. In some experiments, radiolabeled IIa was also detected. Based on the level of photoaffinity labeling of subunits IIo and IIa, relative to their concentration in the transcription reaction, the transcriptional activity of RNA polymerase IIO appears to be greater than 10 times that of IIA. RNA attached to subunits IIo and IIc was estimated to be 16-40 nucleotides in length, whereas RNA attached to subunit IIa was approximately 27-40 nucleotides long. Photoaffinity labeling was sensitive to alpha-amanitin, and required DNA containing an RNA polymerase II promoter.

Adenoviridae↗
PubMed1991

Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II.

We have used a recently developed system that allows the isolation of complexes competent for RNA polymerase II elongation (E. Bengal, A. Goldring, and Y. Aloni, J. Biol. Chem. 264:18926-18932, 1989). Pulse-labeled transcription complexes were formed at the adenovirus major late promoter with use of HeLa cell extracts. Elongation-competent complexes were purified from most of the proteins present in the extract, as well as from loosely bound elongation factors, by high-salt gel filtration chromatography. We found that under these conditions the nascent RNA was displaced from the DNA during elongation. These column-purified complexes were used to analyze the activities of different transcription factors during elongation by RNA polymerase II. We found that transcription factor IIS (TFIIS), TFIIF, and TFIIX affected the efficiency of elongation through the adenovirus major late promoter attenuation site and a synthetic attenuation site composed of eight T residues. These factors have distinct activities that depend on whether they are added before RNA polymerase has reached the attenuation site or at the time when the polymerase is pausing at the attenuation site. TFIIS was found to have antiattenuation activity, while TFIIF and TFIIX stimulated the rate of elongation. In comparison with TFIIF, TFIIS is loosely bound to the elongation complex. We also found that the activities of the factors are dependent on the nature of the attenuator. These results indicate that at least three factors play a major role during elongation by RNA polymerase II.

Adenoviruses, Human↗
PubMed1993

Mechanism of DmS-II-mediated pause suppression by Drosophila RNA polymerase II.

Transcription elongation factor S-II mediates nascent transcript cleavage by RNA polymerase II (Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). We have examined the mechanism of action of the Drosophila S-II analog, DmS-II, in a defined transcription system. Our results show that DmS-II is necessary and sufficient to activate nascent transcript cleavage by RNA polymerase II during transcription of a dC-tailed template. The pattern of transcripts resulting from prolonged action by DmS-II indicates that there are kinetic barriers to transcript shortening. During the cleavage reaction, the polymerase remains in register with the template strand and generates mainly nucleotide dimers. The ability of DmS-II to mediate transcript shortening resides in the carboxyl-terminal half of the protein. Our results support a model for pause suppression in which DmS-II binds to the paused polymerase, causes one cleavage event and is then released from the complex. Elongation by the polymerase then allows a second encounter with the pause site and a second chance of passing the site. Complete pause suppression may require multiple transcript shortening events for some polymerase molecules.

Animals↗
PubMed1978

Large-scale purification and subunit structure of DNA-dependent RNA polymerase II from cauliflower inflorescence.

DNA-dependent RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from cauliflower inflorescence (Brassica oleracae, var. botrytis) was highly purified by polyethyleneimine treatment on a large scale. The solubilized enzyme was partially purified by polyethyleneimine fractionation and subjected to chromatography on DEAE-Sephadex and phosphocellulose, and subsequently to sedimentation in a glycerol gradient. The specific activity (231 nmol/mg per 10 min) of this enzyme was comparable to that reported for other purified eukaryotic RNA polymerases. Analysis of the purified RNA polymerase II by polyacrylamide gel electrophoresis under nondenaturing conditions revealed a single band. The subunit composition of the enzyme was analyzed by electrophoresis under denaturing conditions. The RNA polymerase II contained subunits with molecular weights and molar ratios (in parentheses) of 180 000(1), 130 000(2), 48 000(2), 25 000(4), and 19 500(4).

Centrifugation, Density Gradient↗
PubMed2006

Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155.

Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem-loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications.

Animals↗
PubMed2003

Fate of RNA polymerase II stalled at a cisplatin lesion.

Elongating RNA polymerase II blocked by DNA damage in the transcribed DNA strand is thought to initiate the transcription-coupled repair process. The objective of this study is to better understand the sequence of events that occurs during repair from the time RNA polymerase II first encounters the lesion. This study establishes that an immobilized DNA template containing a unique cisplatin lesion can serve as an in vitro substrate for both transcription and DNA repair. RNA polymerase II is quantitatively stalled at the cisplatin lesion during transcription and can be released from the template, along with the nascent transcript, in an ATP-dependent manner. RNA polymerase II stalled at a lesion and containing a dephosphorylated repetitive carboxyl-terminal domain (CTD) appears to be more sensitive toward release. However, a dephosphorylated CTD can become readily phosphorylated in front of the lesion by CTD kinases in the presence of ATP. The observation that RNA polymerase II and transcript release occurs in a TFIIH-deficient repair extract but not in a reconstituted repair system demonstrates that disassembly of the elongation complex can occur independently of the repair process and vice versa. Indeed, the presence of RNA polymerase II at the lesion does not prevent dual incision from occurring. Finally, we also propose that the Cockayne's syndrome B protein factor, believed to be the mammalian transcription repair coupling factor, is neither involved in transcript release nor required for dual incision in the presence of lesionstalled RNA polymerase II in vitro. More likely, it prevents RNA polymerase from backing up when it encounters the lesion. The ability to transcribe and repair the same damaged DNA molecule fixed on beads, along with the fact that the reaction conditions can be freely altered, provides a powerful tool to study the fate of RNA polymerase II blocked on the cisplatin lesion.

Adenosine Triphosphate↗
PubMed1990

Transcription initiated by RNA polymerase II and purified transcription factors from liver. Cooperative action of transcription factors tau and epsilon in initial complex formation.

Synthesis of accurately initiated transcripts has been reconstituted with RNA polymerase II and a set of five transcription factors purified from rat liver. In addition to three previously identified factors alpha, beta gamma, and delta (Conaway, R. C., and Conaway, J. W. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7356-7360), transcription in the reconstituted liver system requires two novel factors designated tau and epsilon. These five transcription factors comprise two functional classes: (i) promoter recognition factors (tau and epsilon), which interact with template DNA to facilitate formation of a stable initial complex that is subsequently recognized and bound by RNA polymerase II, and (ii) RNA chain initiation factors (alpha, beta gamma, and delta), which do not participate in formation of the initial complex, but which are essential for transcription initiation.

Animals↗
PubMed1990

Transcription initiated by RNA polymerase II and purified transcription factors from liver. Transcription factors alpha, beta gamma, and delta promote formation of intermediates in assembly of the functional preinitiation complex.

Accurate initiation at promoters by RNA polymerase II in a highly purified transcription system from rat liver depends on five accessory factors, which comprise two functional classes: (i) "promoter recognition" factors, designated tau and epsilon, which interact with template DNA to form an initial complex that serves as a recognition site for binding by RNA polymerase II and (ii) "RNA chain initiation" factors, designated alpha, beta gamma, and delta, which do not participate in initial complex formation, but which are essential for initiation (Conaway, J. W., Reines, D., and Conaway, R. C. (1990) J. Biol. Chem. 265, 7552-7558). Here we investigate the roles of alpha, beta gamma, and delta in accurate initiation. Kinetic evidence indicates that all three factors act in a stage prior to RNA synthesis to facilitate formation of a functional preinitiation complex. Moreover, results of "template challenge" experiments argue that all three factors become stably associated with the preinitiation complex during this stage. Neither alpha, beta gamma, nor delta functions catalytically in this process; instead, each factor appears to interact directly and stoichiometrically with intermediates in assembly of the preinitiation complex. Order of addition experiments reveal that transcription factors alpha and beta gamma assemble into the preinitiation complex by an "ordered" mechanism. We discuss two recently proposed models for assembly of the functional preinitiation complex and argue that our findings provide a plausible means of reconciling them.

Animals↗
PubMed1989

Common mechanisms of promoter recognition by RNA polymerases II and III.

Recent results indicate that RNA polymerase III can use upstream promoters that are structurally and functionally very similar to those recognized by RNA polymerase II. The demonstration that RNA polymerases II and III can use the same transcription factors emphasizes the fundamental similarities between these distinct activities. It is also clear now that transcription factors can be functionally interchanged between distantly related species, indicating that the basic structures involved in promoter recognition are highly conserved throughout evolution.

Base Sequence↗
PubMed1978

DNA-dependent RNA polymerase II from Acanthamoeba castellanii. Comparison of the catalytic properties and subunit architectures of the trophozoite and cyst enzymes.

The actively growing cells (trophozoites) of the amoeba Acanthamoeba castellanii were found to contain three or perhaps four different forms of class II DNA-dependent RNA polymerase (EC 2.7.7.6). The chromatographic and catalytic properties of all forms of the Acanthamoeba class II polymerases suggest them to be cognates of the class II polymerases previously reported. The predominant form was purified to near homogeneity and its subunit composition determined. Nine different polypeptides were found associated with the purified enzyme: 21 000; 185 000; 140 000; 70 000; 35 000; 21 000; 19 000; 18 500 and 16 200. These polypeptides were interpreted in terms of two class II RNA polymerases which differ in the molecular weight of their largest subunit. When A. castellanii is transferred to a medium lacking nutrients, the cells undergo cellular differentiation resulting in the formation of metabolically inactive cells (cyst formation). During this process there are significant changes in the RNA sequences transcribed. In contrast to this, we find that the chromatographic and catalytic properties of all of the class II RNA polymerases remain unchanged. Further, the subunit architecture of the predominant form(s) of polymerase II is unaltered. These findings suggest that although new RNA sequences are transcribed during encystment their appearance is not a consequence of extensive alterations in the subunit composition of the major class II RNA polymerase.

Amanitins↗
PubMed1992

Reconstitution of transcription with five purified initiation factors and RNA polymerase II from Saccharomyces cerevisiae.

Yeast RNA polymerase II initiation factors a, b, e, and g were isolated from whole cell extract and found to be sufficient, when combined with bacterially expressed yeast transcription factor (TF) IID, to enable RNA polymerase II to utilize nine different eukaryotic promoters in vitro, and to initiate transcription at sites used in vivo. The purified factors did not contain the previously described transcription factor IIA (TFIIA). TFIIA failed to substitute for any purified factor or to stimulate transcription with the complete set of factors, indicating that its function in crude extracts is primarily as an anti-inhibitor.

Antibodies, Monoclonal↗
PubMed1989

Intragenic and extragenic suppressors of mutations in the heptapeptide repeat domain of Saccharomyces cerevisiae RNA polymerase II.

The largest subunit of RNA polymerase II contains a repeated heptapeptide sequence at its carboxy terminus. Yeast mutants with certain partial deletions of the carboxy-terminal repeat (CTR) domain are temperature-sensitive, cold-sensitive and are inositol auxotrophs. Intragenic and extragenic suppressors of the cold-sensitive phenotype of CTR domain deletion mutants were isolated and studied to investigate the function of this domain. Two types of intragenic suppressing mutations suppress the temperature-sensitivity, cold-sensitivity and inositol auxotrophy of CTR domain deletion mutants. Most intragenic mutations enlarge the repeat domain by duplicating various portions of the repeat coding sequence. Other intragenic suppressing mutations are point mutations in a conserved segment of the large subunit. An extragenic suppressing mutation (SRB2-1) was isolated that strongly suppresses the conditional and auxotrophic phenotypes of CTR domain mutations. The SRB2 gene was isolated and mapped, and an SRB2 partial deletion mutation (srb2 delta 10) was constructed. The srb2 delta 10 mutants are temperature-sensitive, cold-sensitive and are inositol auxotrophs. These phenotypes are characteristic of mutations in genes encoding components of the transcription apparatus. We propose that the SRB2 gene encodes a factor that is involved in RNA synthesis and may interact with the CTR domain of the large subunit of RNA polymerase II.

Amino Acid Sequence↗