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 U6 small nuclear (sn)RNA gene (SNR6) from the yeast Saccharomyces cerevisiae is transcribed by RNA polymerase III in vivo. This gene is unusual in having a TATA box at position -30, and an essential B-block element located downstream of the T-rich termination signal. The B block is one of the two intragenic promoter elements of transfer RNA genes that are recognized by transcription factor (TF)IIIC (ref. 4). But accurate in vitro transcription of yeast U6 snRNA gene by PolIII in a purified system requires only TFIIIB components, including the TATA-box binding protein TBP. Here we report that, after nucleosome reconstitution or chromatin assembly, U6 snRNA synthesis becomes dependent on TFIIIC and on the integrity of the B-block element. This observation resolves an apparent paradox between in vitro and in vivo results concerning the necessity of the downstream B-block element and sheds light on a new role of TFIIIC in gene activation.
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PMID:TFIIIC relieves repression of U6 snRNA transcription by chromatin. 846 80

The protein TFIIB is a general transcription initiation factor that interacts with a promoter complex (D.DNA) containing the TATA-binding subunit (TFIID tau, or TBP) of TFIID to facilitate subsequent interaction with RNA polymerase II (ref. 2) through the associated TFIIF (ref. 3). The potential bridging function of TFIIB raises the possibility of two structural domains and emphasizes the importance of TFIIB structure-function studies for a further understanding of preinitiation complex assembly and function. Here we show that human TFIIB (refs 5,6) is comprised of functionally distinct N- and C-terminal domains. The C-terminal domain, containing the direct repeats and associated basic regions, is necessary and sufficient for interaction with the D.DNA complex. By contrast, the N-terminal domain that is dispensable for formation of the TFIID tau-TFIIB-promoter (D.B.DNA) complex is required for subsequent events leading to basal transcription initiation. On the basis of these results, we discuss structural and functional similarities between TFIIB and TFIID tau, which have similar structural organization and motifs.
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PMID:Functional dissection of TFIIB domains required for TFIIB-TFIID-promoter complex formation and basal transcription activity. 851 7

Simian virus 40 (SV40) large T antigen (Tag) is a promiscuous transcriptional transactivator; however, its mechanism of transactivation remains unknown. Recent studies have suggested the possible involvement of protein-protein interactions with TBP, the TATA box-binding protein of TFIID, and TEF-1, an enhancer-binding factor. We show here that (i) the Tag domain containing amino acids 133 to 249 directly interacts with the general transcription factor TFIIB, the activator protein Sp1, and the 140-kDa subunit of RNA polymerase II, as well as with TBP and TEF-1; (ii) these interactions can also occur when these transcription factors are present in their functional states in cellular extracts; (iii) binding of Tag to TBP is eliminated by preincubation of TBP either at 48 degrees C or with the adenovirus 13S E1a protein; (iv) this domain of Tag cannot bind concurrently to more than one of these transcription factors; and (v) the substitution of Tag amino acid residues 173 and 174 inactivates the ability of this Tag domain both to associate with any of these transcription factors and to transactivate the SV40 late promoter. Thus, we conclude that SV40 Tag probably does not transactivate via the concurrent interaction with multiple components of the preinitiation complex. Rather, we hypothesize that transactivation by Tag may primarily occur by removing or preventing the binding of factors that inhibit the formation of preinitiation complexes.
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PMID:The major transcriptional transactivation domain of simian virus 40 large T antigen associates nonconcurrently with multiple components of the transcriptional preinitiation complex. 855 80

The universal TATA-binding protein, TBP, is an essential component of the multiprotein complex known as transcription factor IID (TFIID). This complex, which consists of TBP and TBP-associated factors (TAFs), is essential for RNA polymerase II-mediated transcription. The molecular size of human TBP (37.7 kD) is close to the passive diffusion limit along the transport channel of the nuclear pore complex (NPC). Therefore, the possibility exists that NPCs restrict TBP translocation to the nuclear interior. Here we show for the first time, with patch-clamp and atomic force microscopy (AFM), that NPCs regulate TBP movement into the nucleus and that TBP (10(-15)-10(-10)M) is capable of modifying NPC structure and function. The translocation of TBP was ATP-dependent and could be detected as a transient plugging of the NPC channels, with a concomitant transient reduction in single NPC channel conductance, gamma, to a negligible value. NPC unplugging was accompanied by permanent channel opening at concentrations greater than 250 pM. AFM images demonstrated that the TBP molecules attached to and accumulated on the NPC cytosolic side. NPC channel activity could be recorded for more than 48 hr. These observations suggest that three novel functions of TBP are: to stabilize NPC, to force the NPC channels into an open state, and to increase the number of functional channels. Since TBP is a major component of transcription, our observations are relevant to the understanding of the gene expression mechanisms underlying normal and pathological cell structure and function.
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PMID:Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex. 856 41

Class III genes depend on TFIIIB for recruitment of RNA polymerase III. Yeast TFIIIB is comprised of three components: TBP, TFIIIB70 and a 90 kDa polypeptide contained in the fraction B". We report the isolation of the yeast gene TFC7 which, based on genetic and biochemical evidence, encodes the 90 kDa polypeptide. TFC7 was isolated as a multicopy suppressor of temperature-sensitive mutations in the two largest subunits of TFIIIC. It is an essential gene, encoding a polypeptide of 68 kDa migrating with an apparent size of approximately 90 kDa. In gel shift assays, recombinant TFC7 protein (rTFC7) alone did not bind detectably to DNA, or to the TFIIIC-DNA complex even in the presence of TBP or TFIIIB70, but it was required to assemble the TFIIIB-TFIIIC-DNA complex. The two-hybrid assay pointed to an interaction between TFC7 protein and tau 131, the second largest subunit of TFIIIC (that also interacts with TFIIIB70). rTFC7p can replace the B" component of TFIIIB for synthesis of U6 RNA in a system reconstituted with recombinant TBP and TFIIIB70 polypeptides and highly purified RNA polymerase III. Surprisingly, specific transcription of the SUP4 tRNATyr gene promoted by rTFC7p was much weaker than with B". An additional factor activity, provided by the recently identified TFIIIE fraction, was required to restore control levels of transcription.
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PMID:A suppressor of mutations in the class III transcription system encodes a component of yeast TFIIIB. 861 41

A requirement for an ATP cofactor in synthesis of the first 8-10 bonds of promoter-specific transcripts by RNA polymerase II is well established. Whether ATP is required for synthesis of the first phosphodiester bond or at a slightly later stage in synthesis of nascent transcripts, however, remains controversial. Goodrich and Tjian (Goodrich, J.A., and Tjian, R. (1994) Cell 77, 145-156) recently proposed that synthesis of the first phosphodiester bond of promoter-specific transcripts by RNA polymerase II is independent of ATP and general transcription factors TFIIE and TFIIH. Here we investigate this model. Taken together, our findings indicate that ATP, TFIIE, and TFIIH can have a profound effect on the efficiency of transcription initiation. First, we observe that synthesis of the first phosphodiester bond of transcripts initiated at the adenovirus 2 major late promoter depends strongly on ATP, TFIIE, and TFIIH in a transcription system reconstituted with RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE, and TFIIF. Second, we demonstrate that, in this enzyme system, ATP-dependent activation of transcription initiation can occur immediately prior to synthesis of the first phosphodiester bond of nascent transcripts. Finally, we demonstrate that the activated initiation complex is unstable and decays rapidly to an inactive state in the presence of the inhibitor ATP-gammaS (adenosine 5'-O-(thio)triphosphate), even during reiterative synthesis of abortive transcripts.
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PMID:A role for ATP and TFIIH in activation of the RNA polymerase II preinitiation complex prior to transcription initiation. 863 33

An intrinsic property of class I gene transcription by RNA polymerase I (Pol I) is the species specificity of the initiation reaction. Previous studies have demonstrated that species-specific rDNA promoter recognition is brought about by a TBP-TAF complex, termed TIF-IB in mouse and SL1 in man. We have compared the ability of affinity-purified TIF-IB and SL1 to direct transcription from the homologous rDNA template both in a reconstituted transcription system and in nuclear extracts prepared from mouse and human cells. We show that Pol I from both species and the individual transcription factors, with the exception of TIF-IB/SL1, are functionally interchangeable in the reconstituted transcription system containing purified proteins. In nuclear extracts, however, species-specific differences are obvious. Whereas SL1 reprograms a heterologous mouse extract to recognize the human promoter, TIF-IB fails to reprogram a human extract unless it is complemented with mouse Pol I. Crude human, but not mouse, Pol I exhibits species-specific differences that disappear after purification. We propose that in extracts and less purified fractions human Pol I exists as 'holoenzyme' containing associated protein(s) that prevent assembly of TIF-IB-directed initiation complexes at the murine rDNA promoter.
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PMID:Species specificity of ribosomal gene transcription: a factor associated with human RNA polymerase I prevents transcription of mouse rDNA. 863 44

ICP4 of herpes simplex virus is responsible for the activation of viral transcription during infection. It also efficiently activates and represses transcription in vitro depending on the promoter context. The contacts made between ICP4 and the cellular proteins that result in activated transcription have not been identified. The inability of ICP4 to activate transcription with TATA-binding protein in place of TFIID and the requirement for an initiator element for efficient ICP-4-activated transcription suggest that coactivators, such as TBP-associated factors, are involved (B. Gu and N. DeLuca, J. Virol. 68:7953-7965, 1994). In this study we showed that ICP4 activates transcription in vitro using an immunopurified TFIID, indicating that TBP-associated factors may be a sufficient subset of coactivators for ICP4-activated transcription. Similar to results seen in vivo, the presence of the ICP4 C-terminal region (amino acids 774 to 1298) was important for activation in vitro. With epitope-tagged ICP4 molecules in immunoaffinity experiments, it was shown that the C-terminal region was also required for ICP4 to interact with TFIID present in a crude transcription factor fraction. In the same assay, ICP4 was unable to interact with the basal transcription factors, TFIIB, TFIIE, TFIIF, and TFIIH and RNA polymerase II. ICP4 could also interact with TBP, independent of the C-terminal region. However, reflective of the interaction between ICP4 and TFIID, the ICP4 C-terminal region was required for an interaction with FAF250-TBP complexes and with TAF250 alone. Therefore, the interfaces or conformation of TBP mediating the interaction between ICP4 and TBP in solution is probably masked when TBP is bound to TAF250. With a series of mutant ICP4 molecules purified from herpes simplex virus-infected cells, it was shown that ICP4 molecules that can bind DNA and interact with TAF250 could activate transcription. Taken together, these results demonstrate that ICP4 interaction with TFIID involves the TAF250 molecule and the C-terminal region of ICP4 and that this interaction is part of the mechanism by which ICP4 activates transcription.
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PMID:Interaction of the viral activator protein ICP4 with TFIID through TAF250. 864 20

We describe a fractionation and purification scheme for the Drosophila RNA polymerase II general transcription factors. Drosophila TFIIE, TFIIF, TFIIH, and RNA polymerase II have been purified to greater than 50% homogeneity from Drosophila embryo nuclear extracts. TFIID has been purified 80-fold and is not significantly contaminated with any of the other general factors. This is the first reported identification and purification of Drosophila TFIIH and TFIIE. Further analysis shows that, similar to their mammalian counterparts, Drosophila TFIIH is composed of eight polypeptides sized between 30 and 100 kDa, and Drosophila TFIIE is composed of two polypeptides sized at 34 and 60 kDa. When all of these fractions are combined with recombinant Drosophila TFlIB, a highly purified in vitro transcription system is generated that has not previously been available in Drosophila. The TFIID fraction can be replaced with recombinant Drosophila TBP to give a transcription system that is nearly free of contaminating proteins.
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PMID:Purification of the Drosophila RNA polymerase II general transcription factors. 865 Jan 70

Contact between a transcriptional activator and one or more components of the RNA polymerase II transcription initiation machinery is generally believed important for activators to function. Several different molecular targets have been suggested for direct contact by herpes simplex virus virion protein VP16, including the general initiation factor TFIIB. In this report we have used several strategies to critically assess this interaction between VP16 and TFIIB. Affinity columns of VP16 bound TFIIB activity from HeLa cell extracts and the binding was reduced by mutations in the activation domain of VP16. In assays of direct binding, VP16 bound recombinant human TFIIB but not Drosophila or yeast TFIIB. Unlike binding from an extract, however, we found that the interaction between VP16 and recombinant human TFIIB was not affected by mutations in VP16 that reduce transactivation. Point mutations within human TFIIB that reduce transactivation by VP16 have been shown to reduce VP16 binding, but we show here that these same mutations critically affect both the important TBP-TFIIB interaction and the ability of TFIIB to support activator-independent basal transcription in vitro. Taken together our results suggest more evidence is needed to support the notion that TFIIB is a functionally important target for the activator VP16.
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PMID:Characterization of the interaction between the acidic activation domain of VP16 and the RNA polymerase II initiation factor TFIIB. 871 May 3


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