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)

Each cycle of transcription appears to be associated with the reversible phosphorylation of the repetitive COOH-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit. The dephosphorylation of RNAP II by CTD phosphatase, therefore, plays an important role in the transcription cycle. The following studies characterize the activity of HeLa cell CTD phosphatase with a special emphasis on the regulation of CTD phosphatase activity. Results presented here suggest that RNAP II contains a docking site for CTD phosphatase that is essential in the dephosphorylation reaction and is distinct from the CTD. This is supported by the observations that (a) phosphorylated recombinant CTD is not a substrate for CTD phosphatase, (b) RNAP IIB, which lacks the CTD, and RNAP IIA are competitive inhibitors of CTD phosphatase and (c) CTD phosphatase can form a stable complex with RNAP II. To test the possibility that the general transcription factors may be involved in the regulation of CTD phosphatase, CTD phosphatase activity was examined in the presence of recombinant or highly purified general transcription factors. TFIIF stimulates CTD phosphatase activity 5-fold. The RAP74 subunit of TFIIF alone contained the stimulatory activity and the minimal region sufficient for stimulation corresponds to COOH-terminal residues 358-517. TFIIB inhibits the stimulatory activity of TFIIF but has no effect on CTD phosphatase activity in the absence of TFIIF. The potential importance of the docking site on RNAP II and the effect of TFIIF and TFIIB in regulating the dephosphorylation of RNAP II at specific times in the transcription cycle are discussed.
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PMID:The activity of COOH-terminal domain phosphatase is regulated by a docking site on RNA polymerase II and by the general transcription factors IIF and IIB. 779 76

To explore the diversity in the mechanisms of basal transcription by RNA polymerase II, we have employed a novel biochemical approach that involves perturbation of the transcription reaction with exogenously added TFIIB or TATA box-binding protein (TBP). Under these conditions, we observe promoter-selective inhibition of transcription by excess TFIIB or excess TBP. This inhibition occurs at the level of basal transcription, because it is observed with minimal promoters that comprise only the TATA box and initiation site sequences as well as with preparations of basal transcription factors that have been purified to greater than 90% homogeneity. In addition, the excess basal factors inhibit the assembly of a functional preinitiation complex but do not inhibit transcription initiation from preassembled preinitiation complexes. A study of several promoters revealed a reciprocal trend in the promoter specificity of inhibition by excess TFIIB versus that by excess TBP. At opposite ends of this spectrum, promoters are strongly inhibited by excess TFIIB but not excess TBP and vice versa. These results reveal the existence of a spectrum of mechanisms for preinitiation complex assembly at different promoters. The mechanistic preference appears to be specified by the aggregate of basal promoter elements rather than by an individual component, such as the TATA box or initiation site sequence. This spectrum provides a new parameter by which differences in the function of minimal class II promoters can be analyzed in the context of both basal and regulated transcription.
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PMID:A spectrum of mechanisms for the assembly of the RNA polymerase II transcription preinitiation complex. 782 20

The diverse functions of Saccharomyces cerevisiae RNA polymerase II are partitioned among its 12 subunits, designated RPB1-RPB12. Although multiple functions have been assigned to the three largest subunits, RPB1, RPB2, and RPB3, the functions of the remaining smaller subunits are unknown. We have determined the function of one of the smaller subunits, RPB9, by demonstrating that it is necessary for accurate start site selection. Transcription in the absence of RPB9 initiates farther upstream at new and previously minor start sites both at the CYC1 promoter in vitro and at the CYC1, ADH1, HIS4, H2B-1, and RPB6 promoters in vivo. Immunoprecipitation of RNA polymerase II from cells lacking the RPB9 gene revealed that all of the remaining 11 subunits are assembled into the enzyme, suggesting that the start site defect is attributable solely to the absence of RPB9. In support of this hypothesis, we have shown that addition of wild-type recombinant RPB9 completely corrects for the start site defect seen in vitro. A mutated recombinant RPB9 protein, with an alteration in a metal-binding domain required for high temperature growth and accurate start site selection in vivo, was at least 10-fold less effective at correcting the start site defect in vitro. RPB9 appears to play a unique role in transcription initiation, as the defects revealed in its absence are distinct from those seen with mutants in RNA polymerase subunit RPB1 and factor e (TFIIB), two other yeast proteins also involved in start site selection.
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PMID:RNA polymerase II subunit RPB9 is required for accurate start site selection. 788 69

Core promoters for RNA polymerase II frequently contain either (or both) of two consensus sequence elements, a TATA box and/or an initiator (Inr). Using test promoters consisting of prototypical TATA and/or Inr elements, together with binding sites for sequence-specific activators, we have analyzed the function of TATA and Inr elements in vivo. In the absence of activators, the TATA element was significantly more active than the Inr, and the combination of elements was only slightly more effective than the TATA-only promoter. In the presence of any of several coexpressed activator proteins, the TATA elements was again most active, but here addition of the Inr allowed significant increases in activity, indicating a cooperative interaction between the two elements. An interesting exception was observed with the activator Sp1, which was more effective with the Inr-only promoter, and addition of a TATA box did not enhance activity. Finally, in all cases the TATA plus Inr promoters were found to be partially or completely resistant to the dominant negative effects of a transcription factor TFIIB mutant previously shown to interfere with expression from TATA-only promoters. This result strengthens the conclusion that TATA and Inr elements can cooperate in vivo.
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PMID:Cooperation between core promoter elements influences transcriptional activity in vivo. 789 7

A core promoter element called an initiator (Inr) overlaps the transcription start site of numerous mammalian protein-coding genes. In promoters that lack a TATA box, the Inr is functionally analogous to TATA, in that it is capable of directing basal transcription by RNA polymerase II and of determining the precise site of transcription initiation. In promoters that contain a TATA box, the Inr can greatly enhance promoter strength. Mammalian Inr consensus sequences have been defined through functional studies and sequence comparisons of the start site regions of protein-coding genes. Here, we show that, in a DNase I footprinting assay with synthetic promoters, the purified TATA-binding protein complex TFIID specifically contacted the Inr. The TFIID-Inr interaction relies on the precise nucleotides needed for Inr function. Detection of the interaction was dependent either on a TATA box or on Sp1 bound to upstream sites. Furthermore, recombinant TFIIB appeared to influence the TFIID-Inr interaction, whereas TFIIA stabilized the TFIID-TATA interaction. These results demonstrate that distinct components of TFIID interact with the TATA boxes and Inr elements of core promoters for RNA polymerase II.
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PMID:Direct recognition of initiator elements by a component of the transcription factor IID complex. 792 70

Efforts to understand the impact of core promoter architecture on the mechanism of transcription initiation by RNA polymerase II have been hampered by lack of well defined, reconstituted transcription systems responsive both to efficiently transcribed consensus and near consensus TATA box-containing promoters and to considerably weaker TATA-less promoters. In this report, we investigate the influence of core promoter structure on the mechanism of assembly of the RNA polymerase II preinitiation complex using a highly purified, holoTFIID-dependent transcription system that permits sensitive measurement of transcription initiation from a wide variety of TATA and TATA-less promoters in the absence of transactivators. A direct comparison of the requirements for formation of stable preinitiation intermediates at these promoters led to the discovery that, whereas holoTFIID binds avidly to the consensus TATA- and strong initiator-containing adenovirus major late (AdML) promoter to form the first stable intermediate on the pathway leading to formation of the complete preinitiation complex, it binds poorly not only to TATA-less promoters but also to promoters with consensus or near consensus TATA elements. With the exception of the AdML promoter, formation of stable preinitiation intermediates at each of the promoters tested was found to be strongly dependent on RNA polymerase II, holoTFIID, and TFIIB and was stimulated by TFIIF. Based on these observations, we suggest that RNA polymerase II assembles with many TATA and TATA-less promoters by a common pathway.
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PMID:Role of core promoter structure in assembly of the RNA polymerase II preinitiation complex. A common pathway for formation of preinitiation intermediates at many TATA and TATA-less promoters. 792 83

We report here that the largest subunit of yeast RNA polymerase II contains an acidic domain that is similar to acidic activators of transcription. This domain includes the highly conserved homology box H. A hybrid protein containing this acidic domain fused to the DNA-binding domain of GAL4 is a potent activator of transcription in the yeast Saccharomyces cerevisiae. Interestingly, mutations that reduce the upstream activating activity of this acidic domain also abolish the normal function of RNA polymerase II. Such functional defects can be rescued by the acidic activation domains of VP16 and GAL4 when inserted into the mutant derivatives of RNA polymerase II. We further show that this acidic domain of RNA polymerase II interacts directly with two general transcription factors, the TATA-binding protein and TFIIB, and that the acidic activation domain of VP16 can compete specifically with the acidic domain of the RNA polymerase for these interactions. We discuss the implications of this finding for the mechanisms of transcriptional activation in eucaryotes.
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PMID:A highly conserved domain of RNA polymerase II shares a functional element with acidic activation domains of upstream transcription factors. 793 66

The human general transcription factor IIF (TFIIF) is required for an accurate transcription initiation by RNA polymerase II and shares some analogous features with the sigma subunit of bacterial RNA polymerase. As an attempt to analyze the function of TFIIF, we examined its effect on bacterial transcription in vitro. TFIIF significantly enhanced the initiation of transcription by the bacterial RNA polymerase while other general transcription factors, TATA-binding protein, TFIIB, and TFIIE, did not. The enhancement of the bacterial transcription was ascribed to the 74 kDa subunit of TFIIF (RAP74). RAP74 had an activity of enhancing the binding of the bacterial RNA polymerase to the promoter. The enhancing activity of RAP74 depended on a low molar ratio of the RNA polymerase to the template DNA. The action of RAP74 in the bacterial transcription may be related to a possible regulatory role of RAP74 in the eukaryotic transcription initiation.
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PMID:Enhancement of bacterial transcription initiation in vitro by the 74 kDa subunit of human general transcription factor IIF (RAP74). 794 16

To further elucidate the mechanism of transcriptional initiation, we used synthetic oligonucleotides to prepare templates containing heteroduplex regions of varying size and location along the DNA of the adenovirus major late promoter. Unlike closed, linear DNA, or DNA with a downstream mismatch, DNA with a mismatch upstream of the initiation site only required the general factors TATA box-binding protein and transcription factor (TF) IIB to direct specific and accurate initiation in vitro by calf thymus RNA polymerase II. In the presence of TFIIF, initiation was possible on closed, linear DNA, but an upstream mismatch region still stimulated transcriptional initiation by more than 100-fold, leading to production of approximately 0.5 transcript/template in the absence of TFIIE, TFIIH, or ATP. The presence of a DNA mismatch was most effective in the -9 to -1 region; furthermore, stimulation by a mismatch did not require that the initiation site be included in the heteroduplex region. Efficient initiation at the immunoglobulin heavy chain promoter in the presence of TATA box-binding protein and TFIIB was also achieved when a mismatch region was introduced from -9 to +3. Our results suggest that initiation by RNA polymerase II in the absence of transcriptional activation is limited by melting of the promoter DNA upstream of the initiation site.
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PMID:Initiation of transcription by RNA polymerase II is limited by melting of the promoter DNA in the region immediately upstream of the initiation site. 798 11

Regulation of transcription by RNA polymerase II in eukaryotic cells requires both basal and accessory factors, which interact through specific protein-DNA or protein-protein interactions. The high mobility group 1 protein (HMG1) was previously demonstrated to be a nonhistone chromatin-associated protein, which selectively recognizes cruciform DNA rather than a specific primary sequence element. During our investigations of proteins that interact with TFIID, we found that purified mammalian HMG1, as well as recombinant human HMG1, can interact with TATA-binding protein (TBP) in the presence of a TATA box-containing oligonucleotide to form a specific HMG1.TBP.promoter complex. This complex prevents TFIIB binding to TBP and consequently blocks formation of the preinitiation complex. In contrast, TFIIA can compete with HMG1 for binding to TBP. In an in vitro transcription assay reconstituted with highly purified or recombinant general factors, HMG1 is able to inhibit transcription by RNA polymerase II over 30-fold. As expected, addition of TFIIA can partially reverse this repression in a concentration-dependent manner. These results demonstrate that HMG1, a chromatin-associated protein, has the potential to act as a TBP-dependent negative transcription factor and may provide an important link between chromatin structure and the modulation of class II gene transcription.
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PMID:The high mobility group protein HMG1 can reversibly inhibit class II gene transcription by interaction with the TATA-binding protein. 800 19


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