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PubMed1995

The cellular factor TRP-185 regulates RNA polymerase II binding to HIV-1 TAR RNA.

Activation of HIV-1 gene expression by the transactivator Tat is dependent on an RNA regulatory element located downstream of the transcription initiation site known as TAR. To characterize cellular factors that bind to TAR RNA and are involved in the regulation of HIV-1 transcription, HeLa nuclear extract was fractionated and RNA gel-retardation analysis was performed. This analysis indicated that only two cellular factors, RNA polymerase II and the previously characterized TAR RNA loop binding protein TRP-185, were capable of binding specifically to TAR RNA. To elucidate the function of TRP-185, it was purified from HeLa nuclear extract, amino acid microsequence analysis was performed and a cDNA encoding TRP-185 was isolated. TRP-185 is a novel protein of 1621 amino acids which contains a leucine zipper and potentially a novel RNA binding motif. In gel-retardation assays, the binding of both recombinant TRP-185 and RNA polymerase II was dependent on the presence of an additional group of proteins designated cellular cofactors. Both the TAR RNA loop and bulge sequences were critical for RNA polymerase II binding, while TRP-185 binding was dependent only on TAR RNA loop sequences. Since binding of TRP-185 and RNA polymerase II to TAR RNA was found to be mutually exclusive, our results suggest that TRP-185 may function either alone or in conjunction with Tat to disengage RNA polymerase II which is stalled upon binding to nascently synthesized TAR RNA during transcriptional elongation.

Amino Acid Sequence↗

PubMed1997

A serine/arginine-rich nuclear matrix cyclophilin interacts with the C-terminal domain of RNA polymerase II.

The largest subunit of RNA polymerase II shows a striking difference in the degree of phosphorylation, depending on its functional state: initiating and elongating polymerases are unphosphorylated and highly phosphorylated respectively. Phosphorylation mostly occurs at the C-terminal domain (CTD), which consists of a repetitive heptapeptide structure. Using the yeast two-hybrid system, we have selected for mammalian proteins that interact with the phosphorylated CTD of mammalian RNA polymerase II. A prominent isolate, designated SRcyp/CASP10, specifically interacts with the CTD not only in vivo but also in vitro . It contains a serine/arginine-rich (SR) domain, similar to that found in the SR protein family of pre-mRNA splicing factors, which is required for interaction with the CTD. Most remarkably, the N-terminal region of SRcyp includes a peptidyl-prolyl cis - trans isomerase domain characteristic of immunophilins/cyclophilins (Cyp), a protein family implicated in protein folding, assembly and transport. SRcyp is a nuclear protein with a characteristic distribution in large irregularly shaped nuclear speckles and co-localizes perfectly with the SR domain-containing splicing factor SC35. Recent independent investigations have provided complementary data, such as an association of the phosphorylated form of RNA polymerase II with the nuclear speckles, impaired splicing in a CTD deletion background and inhibition of in vitro splicing by CTD peptides. Taken together, these data indicate that factors directly or indirectly involved in splicing are associated with the elongating RNA polymerases, from where they might translocate to the nascent transcripts to ensure efficient splicing, concomitant with transcription.

Amino Acid Isomerases↗

PubMed1992

Recombinant TBP, transcription factor IIB, and RAP30 are sufficient for promoter recognition by mammalian RNA polymerase II.

Initiation of transcription by RNA polymerase II is a complex, multistep process which requires several accessory factors in addition to the polymerase itself. A critical event in transcription initiation is the specific association of RNA polymerase II with promoter DNA. In this report we show that three eukaryotic polypeptides, produced in Escherichia coli and purified to near homogeneity, constitute a minimal set of general transcription factors both necessary and sufficient for specific and stable promoter binding by RNA polymerase II. These polypeptides are the yeast TATA box binding protein TBP, the human general initiation factor TFIIB, and human RAP30, the small subunit of RAP30/74 (or transcription factor IIF). Formation of the polymerase-containing complex required only the TATA box, and not the initiator element (Inr), of the adenovirus major late promoter which was used in these experiments.

Base Sequence↗

PubMed2004

Newly Initiated RNA encounters a factor involved in splicing immediately upon emerging from within RNA polymerase II.

We employed RNA-protein cross-linking to map the path of the nascent RNA as it emerges from within RNA polymerase II. A UV-cross-linkable uridine analog was incorporated at two positions within the first five nucleotides of the transcript. Only the two largest subunits of RNA polymerase II cross-linked to the transcript in complexes containing 17-24-nucleotide (nt) RNAs. Extension of the RNA to 26 or 28 nt revealed an additional strong cross-link to the splicing factor U2AF65. In U17 complexes, in which the RNA is still contained within the polymerase, U2AF65 is tightly bound. In contrast, U2AF65 is more loosely bound in C28 transcription complexes, in which about 10 nt of transcript have emerged from the RNA polymerase. Cross-linking of U2AF65 to RNA in a C28 complex was eliminated by the addition of an excess of an RNA oligonucleotide containing the consensus U2AF65 binding site, but U2AF65 was not displaced by a nonconsensus RNA. These findings indicate that U2AF65 shifts from protein-protein to protein-RNA interactions as the RNA emerges from the polymerase. During transcription of one particular template at low UTP concentration, RNA polymerase II pauses just after synthesizing a transcript segment that is a U2AF65 binding site. Dwell time of the polymerase at this pause site was significantly and specifically reduced by the addition of recombinant U2AF65 to the transcription reaction. Therefore, the association of U2AF65 with RNA polymerase II may function not only to deliver U2AF65 to the nascent transcript but also to modulate efficient transcript elongation.

Cell Nucleus↗

PubMed1999

The general transcription factors IIA, IIB, IIF, and IIE are required for RNA polymerase II transcription from the human U1 small nuclear RNA promoter.

RNA polymerase II transcribes the mRNA-encoding genes and the majority of the small nuclear RNA (snRNA) genes. The formation of a minimal functional transcription initiation complex on a TATA-box-containing mRNA promoter has been well characterized and involves the ordered assembly of a number of general transcription factors (GTFs), all of which have been either cloned or purified to near homogeneity. In the human RNA polymerase II snRNA promoters, a single element, the proximal sequence element (PSE), is sufficient to direct basal levels of transcription in vitro. The PSE is recognized by the basal transcription complex SNAPc. SNAPc, which is not required for transcription from mRNA-type RNA polymerase II promoters such as the adenovirus type 2 major late (Ad2ML) promoter, is thought to recruit TATA binding protein (TBP) and nucleate the assembly of the snRNA transcription initiation complex, but little is known about which GTFs other than TBP are required. Here we show that the GTFs IIA, IIB, IIF, and IIE are required for efficient RNA polymerase II transcription from snRNA promoters. Thus, although the factors that recognize the core elements of RNA polymerase II mRNA and snRNA-type promoters differ, they mediate the recruitment of many common GTFs.

Animals↗

PubMed1995

Both RNA polymerase III and RNA polymerase II accurately initiate transcription from a human U6 promoter in vitro.

The promoter of vertebrate U6 small nuclear RNA genes consists of a TATA box and a snRNA proximal sequence element (PSE), and the combination of these two elements directs RNA polymerase III transcription. We detected RNA polymerase II transcription as well as pol III transcription from the human U6 promoter in a HeLa nuclear extract. The pol II-specific transcription was independent of the PSE and dependent on the presence of the TATA box. Both pol III- and pol II-specific transcription were stimulated by addition of recombinant TATA-binding protein (TBP). We conclude that both pol III and pol II preinitiation complexes can assemble on the U6 promoter in vitro and could compete during the bona fide process in the cell.

Animals↗

PubMed1998

Molecular genetics of the RNA polymerase II general transcriptional machinery.

Transcription initiation by RNA polymerase II (RNA pol II) requires interaction between cis-acting promoter elements and trans-acting factors. The eukaryotic promoter consists of core elements, which include the TATA box and other DNA sequences that define transcription start sites, and regulatory elements, which either enhance or repress transcription in a gene-specific manner. The core promoter is the site for assembly of the transcription preinitiation complex, which includes RNA pol II and the general transcription fctors TBP, TFIIB, TFIIE, TFIIF, and TFIIH. Regulatory elements bind gene-specific factors, which affect the rate of transcription by interacting, either directly or indirectly, with components of the general transcriptional machinery. A third class of transcription factors, termed coactivators, is not required for basal transcription in vitro but often mediates activation by a broad spectrum of activators. Accordingly, coactivators are neither gene-specific nor general transcription factors, although gene-specific coactivators have been described in metazoan systems. Transcriptional repressors include both gene-specific and general factors. Similar to coactivators, general transcriptional repressors affect the expression of a broad spectrum of genes yet do not repress all genes. General repressors either act through the core transcriptional machinery or are histone related and presumably affect chromatin function. This review focuses on the global effectors of RNA polymerase II transcription in yeast, including the general transcription factors, the coactivators, and the general repressors. Emphasis is placed on the role that yeast genetics has played in identifying these factors and their associated functions.

RNA Polymerase II↗

PubMed1981

Transcription of globin genes in murine erythroleukemic cell chromatin by RNA polymerase II from mouse cells.

RNA polymerase II was purified from mouse Ehrlich ascites tumor cells, and was used to transcribe chromatin from Friend leukemic cells at various stages of induction. The transcription was entirely dependent on exogenous RNA polymerase, and was inhibited by actinomycin D, or a low dose of alpha-amanitin. Globin-specific sequences of the transcripts were determined by hybridization of 3H-labeled transcripts with plasmid DNA immobilized on filters. When chromatin from uninduced cells was used, less than 0.01% of the total radioactivity was retained on the globin DNA-filters, whereas about 0.05% was retained when chromatin was made from induced cells. This value was about one-ninth of the content of cytoplasmic globin mRNA which was pulse-labeled in induced cells. The transcription of whole chromatin or globin genes was inhibited almost completely by a rifampicin derivative, but in the case of chromatin, both sense and anti-sense strands of globin were transcribed. From these results, it is suggested that isolated chromatin retains some native conformation but is transcribed aberrantly.

Animals↗

PubMed2005

Nature of the nucleosomal barrier to RNA polymerase II.

In the cell, RNA polymerase II (pol II) efficiently transcribes DNA packaged into nucleosomes, but in vitro encounters with the nucleosomes induce catalytic inactivation (arrest) of the pol II core enzyme. To determine potential mechanisms making nucleosomes transparent to transcription in vivo, we analyzed the nature of the nucleosome-induced arrest. We found that the arrests have been detected mostly at positions of strong intrinsic pause sites of DNA. The transient pausing makes pol II vulnerable to arrest, which involves backtracking of the elongation complex for a considerable distance on DNA. The histone-DNA contacts reestablished in front of pol II stabilize backtracked conformation of the polymerase. In agreement with this mechanism, blocking of backtracking prevents nucleosome-induced arrest. Transcript cleavage factor TFIIS reactivates the backtracked complexes and promotes pol II transcription through the nucleosome. Our findings establish the crucial role of elongation factors that suppress pol II pausing and backtracking for transcription in the context of chromatin.

Base Sequence↗

PubMed1990

RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.

Cell Cycle↗

PubMed1995

Purification and characterization of DNA-dependent RNA polymerase II from Candida utilis.

DNA-dependent RNA polymerase II from Candida utilis has been purified to near homogeneity. The purified enzyme resolved into three subforms, viz. IIO, IIA and IIB. On SDS-PAGE the enzyme showed ten polypeptides with molecular weights in the range of 205 kDa to 14 kDa. By two dimensional electrophoresis (IEF followed by SDS-PAGE) the presence of basic and acidic polypeptides has been demonstrated. The enzyme showed Km values of 5, 5.6 and 8 microM for GTP, CTP and ATP, respectively, and the activity was inhibited by low levels of alpha-amanitin and antibodies raised against bovine RNA polymerase II. By Western blot analysis the enzyme was found to cross-react with antibodies to bovine RNA polymerase II. RNA polymerase II from C. utilis is a phosphoprotein, the subunits RPB1 and RPB10 were found to be phosphorylated. Analysis of carboxy-terminal domain indicated that it was functionally redundant at least in case of non-specific transcription, implicating its role in other nuclear processes, such as promoter specific initiation or transcription activation or RNA processing.

Candida↗

PubMed2000

DSIF and NELF interact with RNA polymerase II elongation complex and HIV-1 Tat stimulates P-TEFb-mediated phosphorylation of RNA polymerase II and DSIF during transcription elongation.

Control of transcription elongation requires a complex interplay between the recently discovered positive transcription elongation factor b (P-TEFb) and negative transcription elongation factors, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) sensitivity inducing factors (DSIF) and the negative elongation factor (NELF). Activation of HIV-1 gene expression is regulated by a nascent RNA structure, termed TAR RNA, in concert with HIV-1 Tat protein and these positive and negative elongation factors. We have used a stepwise RNA pol II walking approach and Western blotting to determine the dynamics of interactions between HIV-1 Tat, DSIF/NELF, and the transcription complexes actively engaged in elongation. In addition, we developed an in vitro kinase assay to determine the phosphorylation status of proteins during elongation stages. Our results demonstrate that DSIF/NELF associates with RNA pol II complexes during early transcription elongation and travels with elongation complexes as the nascent RNA is synthesized. Our results also show that HIV-1 Tat protein stimulated DSIF and RNA pol II phosphorylation by P-TEFb during elongation. These findings reveal a molecular mechanism for the negative and positive regulation of transcriptional elongation at the HIV-1 promoter.

Cell Nucleus↗

PubMed2003

Roles for BTAF1 and Mot1p in dynamics of TATA-binding protein and regulation of RNA polymerase II transcription.

Regulation of RNA polymerase II (pol II) transcription is a highly dynamic process requiring the coordinated interaction of an array of regulatory proteins. Central to this process is the TATA-binding protein (TBP), the key component of the multiprotein complex TFIID. Interaction of TBP with core promoters nucleates the assembly of the preinitiation complex and subsequent recruitment of pol II. Despite recent advances in our understanding of the dynamic nature of the pol II transcription apparatus, the dynamics of TBP function on pol II promoters has remained largely unexplored. Human BTAF1 (TAF(II)170/TAF-172) and its yeast ortholog, Mot1p, are evolutionarily conserved members of the SNF2-like family of ATPase proteins. Genetic identification of Mot1p as a repressor of pol II transcription was supported by findings that Mot1p and BTAF1 could dissociate TBP from TATA DNA complexes using the energy of ATP hydrolysis. Recent data have revealed new aspects of BTAF1 and Mot1p as positive regulators of TBP function in the pol II system and have described new observations relating to their molecular mechanism of action. We review these data in the context of previous findings with particular attention paid to how human BTAF1 and Mot1p may dynamically regulate TBP function on pol II promoters in cells.

Adenosine Triphosphatases↗

PubMed1984

Affinity-purified antibody as a probe of RNA polymerase II subunit structure.

Antibodies reactive against mammalian RNA polymerase II were isolated by affinity chromatography. Serum from a goat immunized with Drosophila RNA polymerase II was passed over a column containing covalently coupled calf thymus RNA polymerase II, and reactive antibodies were eluted at low pH. The affinity-purified antibody was reactive against two large subunits (Mr = 140,000-220,000) and four small subunits (Mr = 16,000-35,000) of RNA polymerase II purified from calf, human, chicken, and Drosophila, as well as two large subunits of wheat germ RNA polymerase II. These findings confirm that RNA polymerase II subunits share conserved antigenic determinants over wide evolutionary distances. The affinity-purified antibody was also used to investigate the presence and structure of RNA polymerase II in human cell extracts capable of carrying out promoter-selective transcription in vitro. The subunit structure of RNA polymerase in the extract appears to be the same as that of purified human RNA polymerase II. In both cases, the human enzyme differs from the calf thymus enzyme in that it contains a large (Mr = 220,000) form of the largest subunit.

Animals↗

PubMed1988

The 24,000 Da subunit is not required for the RNA synthesis activity of chicken leukemia RNA polymerase II.

The partially purified RNA polymerase II from chicken leukemia cells (Chuang R. Y., Chuang L. F. & Israel M. (1986) Biochem. Pharmacol. 35, 1293-1297) contained multiple subunits with molecular masses (in Da) ranging from 220,000 to 24,000, as shown by SDS-polyacrylamide gel electrophoresis. The enzyme was further purified through phosphocellulose column and fractions containing the enzyme activity were collected and concentrated 400-fold through a microconcentrator. The microconcentrator contained a membrane with a molecular weight cutoff around 30,000 and, hence, removed the 24,000 Da polypeptide from the enzyme. It was found that the resulting enzyme retained all the catalytic activity as compared to the enzyme preparation before the concentration step, suggesting that the stoichiometric amount of the 24,000 Da polypeptide is not required for RNA synthesis activity with a denatured DNA template.

Animals↗

PubMed2006

Role for the Ssu72 C-terminal domain phosphatase in RNA polymerase II transcription elongation.

The RNA polymerase II (RNAP II) transcription cycle is accompanied by changes in the phosphorylation status of the C-terminal domain (CTD), a reiterated heptapeptide sequence (Y(1)S(2)P(3)T(4)S(5)P(6)S(7)) present at the C terminus of the largest RNAP II subunit. One of the enzymes involved in this process is Ssu72, a CTD phosphatase with specificity for serine-5-P. Here we report that the ssu72-2-encoded Ssu72-R129A protein is catalytically impaired in vitro and that the ssu72-2 mutant accumulates the serine-5-P form of RNAP II in vivo. An in vitro transcription system derived from the ssu72-2 mutant exhibits impaired elongation efficiency. Mutations in RPB1 and RPB2, the genes encoding the two largest subunits of RNAP II, were identified as suppressors of ssu72-2. The rpb1-1001 suppressor encodes an R1281A replacement, whereas rpb2-1001 encodes an R983G replacement. This information led us to identify the previously defined rpb2-4 and rpb2-10 alleles, which encode catalytically slow forms of RNAP II, as additional suppressors of ssu72-2. Furthermore, deletion of SPT4, which encodes a subunit of the Spt4-Spt5 early elongation complex, also suppresses ssu72-2, whereas the spt5-242 allele is suppressed by rpb2-1001. These results define Ssu72 as a transcription elongation factor. We propose a model in which Ssu72 catalyzes serine-5-P dephosphorylation subsequent to addition of the 7-methylguanosine cap on pre-mRNA in a manner that facilitates the RNAP II transition into the elongation stage of the transcription cycle.

Alanine↗

PubMed1994

Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II.

Using a defined RNA polymerase II (pol II) transcription system, we have investigated the roles of basal factors at discrete stages during the transcription cycle (e.g., initiation, promoter clearance, and transcript elongation). Abortive initiation assays revealed that TATA-binding protein, transcription factors TFIIB and TFIIF, and pol II were necessary and sufficient to form functional initiation complexes on both linear and supercoiled templates. By contrast, TFIIE, TFIIH, and ATP hydrolysis were additionally required during promoter clearance from linear templates, while negative supercoiling obviated the need for these auxiliary factors. Furthermore, TFIIE, TFIIH, and supercoiling were not required during elongation. Our results suggest a role for TFIIH-associated helicase activity or supercoiling during promoter clearance rather than open complex formation. These results establish abortive initiation as a useful assay for studying functional initiation complex formation in defined eukaryotic transcription systems and provide a framework for investigating regulation at different stages of the eukaryotic transcription cycle.

Adenosine Triphosphate↗

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↗