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)

The human general transcription factor TFIIA is one of several factors involved in specific transcription by RNA polymerase II, possibly by regulating the activity of the TATA-binding subunit (TBP) of TFIID. TFIIA purified from HeLa extracts consists of 35-, 19-, and 12-kDa subunits. Here we describe the isolation of a cDNA clone (hTFIIA gamma) encoding the 12-kDa subunit. Using expression constructs derived from hTFIIA gamma and TFIIA alpha/beta (which encodes a 55-kDa precursor to the alpha and beta subunits of natural TFIIA), we have constructed a synthetic TFIIA with a polypeptide composition similar to that of natural TFIIA. The recombinant complex supports the formation of a DNA-TBP-TFIIA complex and mediates both basal and Gal4-VP16-activated transcription by RNA polymerase II in TFIIA-depleted nuclear extracts. In contrast, TFIIA has no effect on tRNA and 5S RNA transcription by RNA polymerase III in this system. We also present evidence that both the p55 and p12 recombinant subunits interact with TBP and that the basic region of TBP is critical for the TFIIA-dependent function of TBP in nuclear extracts.
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PMID:Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription. 772 59

Initiation of ribosomal RNA synthesis by RNA polymerase I requires the promoter selectivity factor SL1, which consists of the TATA-binding protein, TBP, and three associated factors, TAFIS 110, 63, and 48. Here the in vivo and in vitro assembly of functional SL1 complexes from recombinant TAFIS and TBP are reported. Complexes containing TBP and all three TAFIS were as active in supporting transcription from the human ribosomal RNA gene promoter as endogenous SL1, whereas partial complexes without TBP did not efficiently direct transcription in vitro. These results suggest that TAFIS 110, 63, and 48, together with TBP, are necessary and sufficient to reconstitute a transcriptionally active SL1 complex.
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PMID:Assembly of transcriptionally active RNA polymerase I initiation factor SL1 from recombinant subunits. 780 Nov 30

Regions rich in serine, threonine, and proline residues can be found in transcriptional activation domains, as well as in the N-terminal parts of mammalian TATA-binding proteins, where they are interrupted by polyglutamine stretches. Likewise, the C-terminal domain of the largest subunit of RNA polymerase II contains multiple repeats of the consensus heptapeptide sequence YSPTSPS. To test directly for possible activation functions, we fused the GAL4 DNA-binding domain to the N-terminal domain of human TBP or subdomains of it, and to the C-terminal domain (CTD) of mouse RNA polymerase II or synthetic polymers of a CTD consensus repeat. We found that these chimeric proteins were able to activate transcription when bound to a GAL4 site in front of the TATA box, a function characteristic of transcription factors. However, while subdomains of TBP functioned only from a position close to the TATA box ("promoter" position), multiple repeats of the CTD consensus sequence were also able to mediate transcriptional activation from a remote ("enhancer") position. Our findings suggest that a region of TBP that is unique to mammals functionally cooperates with "proximal" activation domains of promoter-bound transcription factors. They also imply that the C-terminal domain of RNA polymerase II includes a function that is otherwise confined to remote activation domains of enhancer-bound transcription factors. We suggest that the CTD of RNA polymerase II contains a "portable" remote activation domain that may also facilitate chromatin opening within the transcription unit.
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PMID:Basal components of the transcription apparatus (RNA polymerase II, TATA-binding protein) contain activation domains: is the repetitive C-terminal domain (CTD) of RNA polymerase II a "portable enhancer domain"? 782 25

The basal factor TFIIE is an important component of the RNA polymerase II transcription machinery. In our efforts to determine the role of TFIIE in the transcription process, we have begun to define the interactions between TFIIE and other basal transcription factors. Here we report that TFIIE binds selectively to the nonphosphorylated form of RNA polymerase II (IIa) and that this interaction is mediated by the 56-kD subunit of TFIIE. Additional binding studies reveal that TFII can interact with TBP as well as TFIID and that this interaction is mediated primarily via the 56-kD subunit. Our studies indicate that TFIIE also interacts with both subunits of TFIIF and with TFIIH, a multisubunit basal factor reported to catalyze RNA polymerase II CTD phosphorylation. Protein affinity assays demonstrate that TFIIE binds directly to ERCC-3, a DNA repair protein associated with TFIIH. More importantly, TFIIE affinity resin can selectively isolate transcriptionally competent TFIIH from a partially purified preparation and thereby may recruit TFIIH to the transcription complex in vivo. These multiple interactions between TFIIE, Pol II and TFIIH support a model in which TFIIE plays a role in promoter clearance as well as in the recruitment of proteins required for transcription-coupled DNA repair.
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PMID:Transcription factor IIE binds preferentially to RNA polymerase IIa and recruits TFIIH: a model for promoter clearance. 792 47

We used mutant yeast and human TBP molecules with an altered DNA-binding specificity to examine the role of TBP in transcriptional activation in vivo. We show that yeast TBP is functionally equivalent to human TBP for response to numerous transcriptional activators in human cells, including those that do not function in yeast. Despite the extensive conservation of TBP, its ability to respond to transcriptional activators in vivo is curiously resistant to clustered sets of alanine substitution mutations in different regions of the protein, including those that disrupt DNA binding and basal transcription in vitro. Combined sets of these mutations, however, can attenuate the in vivo activity of TBP and can differentially affect response to different activation domains. Although the activity of TBP mutants in vivo did not correlate with DNA binding or basal transcription in vitro, it did correlate with binding in vitro to the largest subunit of TFIID, hTAFII250. Together, these data suggest that TBP utilizes multiple interactions across its surface to respond to RNA polymerase II transcriptional activators in vivo; some of these interactions appear to involve recruitment of TBP into TFIID, whereas others are involved in response to specific types of transcriptional activators.
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PMID:Multiple regions of TBP participate in the response to transcriptional activators in vivo. 795 31

A basal repressor of class II gene transcription was identified, purified, and found to be identical to nonhistone chromosomal protein HMG2. HMG2 was shown to inhibit basal transcription under conditions in which transcription templates form soluble complexes with HMG2. Order-of-addition experiments clearly revealed that HMG2 acted after assembly of a TBP-TFIIA-promoter complex and before formation of the fourth phosphodiester bond by RNA polymerase II. Subsequently, an activity that efficiently counteracted repression of transcription by HMG2 in both TBP- and TFIID-containing transcription systems was isolated. Several lines of evidence suggested that antirepression was mediated by a TFIIH-associated factor. The antirepressor first coeluted with TFIIH, was depleted from this fraction by antibodies directed against the TFIIH subunit p62, was dependent on either ATP or dATP, and then was inhibited by the ATP analogs AMP-PNP and ATP gamma S. Relief of HMG2-mediated repression as well as basal promoter function of TFIIH may involve a helicase that coelutes with TFIIH and displays similar nucleotide specificities. Taken together, these data suggest novel consequences of chromatin-associated HMG proteins and they provide direct evidence for a role of TFIIH-associated enzymes in ATP-dependent antirepression of nonhistone chromosomal proteins.
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PMID:Repression of basal transcription by HMG2 is counteracted by TFIIH-associated factors in an ATP-dependent process. 800 73

The past year has provided new insights into the biochemical mechanism of gene activation. Key discoveries include the finding that TFIIA plays an important regulatory role in transcription complex assembly, the TBP-associated factors are direct targets of at least two classes of activator, and a largely pre-assembled transcription complex has been isolated from yeast cells, challenging the step-wise assembly pathway. This review also presents an update on the argument that TFIIB is the target of VP16 and insights into the energetic role of ATP in RNA polymerase II initiation.
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PMID:The role of activators in assembly of RNA polymerase II transcription complexes. 803 1

Yeast transcription factor TFIIIB is a multicomponent factor comprised of the TATA-binding protein TBP and of associated factors TFIIIB70 and B". Epitope-tagged or histidine-tagged TFIIIB70 could be quantitatively removed from TFIIIB by affinity chromatography. TBP and B" (apparent mass 160-200 kDa) could be easily separated by gel filtration or ion-exchange chromatography. While only weak interactions were detected between TBP and B", direct binding of [35S]-labeled TBP to membrane-bound TFIIIB70 could be demonstrated in absence of DNA. On tRNA genes, there was no basal level of transcription in the complete absence of TBP. The two characterized TFIIIB components (recombinant rTFIIIB70 and rTBP) and a fraction cochromatographing with B" activity were found to be required for TFIIIC-independent transcription of the TATA-containing U6 RNA gene in vitro. Therefore, beside the TFIIIC-dependent assembly process, each TFIIIB component must have an essential role in DNA binding or RNA polymerase recruitment.
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PMID:Interactions between yeast TFIIIB components. 751 81

We report the characterization of a mutation affecting tau 138, the largest subunit of yeast transcription factor IIIC (TFIIIC). A previously described thermosensitive mutation (tsv115), tightly linked to the centromere of chromosome I (Harris, S.D., and Pringle, J.R. (1991) Genetics 127, 279-285) is shown to lie in the TFC3 gene which encodes tau 138. The tau 138 subunit carrying this mutation bears a single substitution of Glu for Gly at position 349 (G349E). In extracts from mutant cells, both the level of TFIIIC and its affinity for tDNA were found to be reduced. The tDNA binding activity of mutant TFIIIC protein was very sensitive to mild heat treatments, and TFIIIC-DNA interaction was inhibited at moderate salt concentrations, as evidenced by gel shift assays. In addition, the tsv115 mutation affected 5 S RNA synthesis in vitro, suggesting that the tau 138 subunit also plays a role in recognition of the TFIIIA-5 S DNA complex. Multicopy suppressors of the TFIIIC defect were sought to reveal components participating in TFIIIC function. One class of suppressors encodes known components of the transcription machinery: two TFIIIC subunits, tau 95 and tau 131, the 70-kDa subunit of TFIIIB, TBP, and a shared subunit of RNA polymerase (pol) I, II, and III, ABC10 alpha; it also includes genes potentially related to pol III function, such as SRP40 which also suppresses a mutation in a subunit shared by RNA polymerases I and III. A second class of suppressors is not involved in transcription but alleviates the main physiological defects of mutant cells. It includes RPR1 and NOP1, required for the maturation of pre-tRNA and pre-rRNA, respectively.
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PMID:A mutation in the largest subunit of yeast TFIIIC affects tRNA and 5 S RNA synthesis. Identification of two classes of suppressors. 808 43

A protein--DNA complex containing TFIID has been analyzed by crosslinking. The TBP subunit of TFIID crosslinked to the TATA element but not to any of the regions further downstream which were tested. A 150 kd polypeptide, which corresponds in size to one of the TBP-associated factors (TAFs), crosslinked to a region between +10 and +15 and a second region between +35 and +47. Another polypeptide of greater than 205 kd (also a potential TAF) crosslinked preferentially to the region between +35 and +42. The +10 to +15 region has been recently implicated in hsp70 promoter recognition by TFIID, and the most downstream contacts overlap with the region where RNA polymerase II pauses on the hsp70 promoter in noninduced cells. Crosslinking revealed that as the salt concentration was increased, the TBP interaction was largely unaffected whereas the protein/DNA interactions downstream of the TATA element were disrupted. We propose that during the formation of a transcription complex, TATA-dependent interactions could be disrupted in the vicinity of the start site and the region immediately downstream. A protein contact downstream of +35 might function in pausing polymerase.
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PMID:Protein/DNA crosslinking of a TFIID complex reveals novel interactions downstream of the transcription start. 813 22


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