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 immunodeficiency virus (HIV) encodes a transcriptional transactivator (Tat), which binds to an RNA hairpin called the transactivation response element (TAR) that is located downstream of the site of initiation of viral transcription. Tat stimulates the production of full-length viral transcripts by RNA polymerase II (pol II). In this study, we demonstrate that Tat coimmunoprecipitates with the pol II holoenzyme in cells and that it binds to the purified holoenzyme in vitro. Furthermore, Tat affinity chromatography purifies a holoenzyme from HeLa nuclear extracts which, upon addition of TBP and TFIIB, supports Tat transactivation in vitro, indicating that it contains all the cellular proteins required for the function of Tat. By demonstrating that Tat interacts with the holoenzyme in the absence of TAR, our data suggest a single-step assembly of Tat and the transcription complex on the long terminal repeat of HIV.
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PMID:The human immunodeficiency virus transactivator Tat interacts with the RNA polymerase II holoenzyme. 912 29

SV40 large T antigen is a multifunctional regulatory protein that plays a key role in the viral life cycle and can stimulate cell proliferation. To accomplish this, large T antigen has to control the expression of cellular genes involved in cell cycle progression and cell growth. rRNA synthesis by RNA polymerase I (Pol I) is tightly associated with cell growth and proliferation, and previous studies indicated that large T antigen up-regulates RNA Pol I transcription in SV40-infected cells. How this process occurs is currently unclear. To investigate the mechanisms of large T antigen stimulation of RNA Pol I transcription, we have established an in vitro transcription system that is responsive to large T antigen. Here, we show that recombinant large T antigen stimulates Pol I transcription reconstituted with purified RNA Pol I, UBF, and the TBP/TAF complex SL1. Immunoprecipitation experiments revealed that large T antigen directly binds to SL1, in vitro, as well as in SV40-infected cells. In addition, our data indicate that this interaction occurs by direct association with three SL1 subunits, namely TBP, TAF(I)48, and TAF(I)110. Transcription studies with large T antigen deletion mutants show that the 538-amino-acid amino-terminal domain is necessary for full stimulation of Pol I transcription. Importantly, mutants that no longer bind to SL1 are also unable to stimulate Pol I transcription. This indicates that recruitment of large T antigen to the rRNA promoter by SL1 constitutes a crucial step in the activation process. Taken together with recent studies on large T antigen activation of RNA Pol II transcription, these results suggest that viral modulation of genes involved in cell proliferation involves direct targeting of promoter-specific TBP/TAF complexes (i.e., SL1 or TFIID) by large T antigen.
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PMID:SV40 large T antigen binds to the TBP-TAF(I) complex SL1 and coactivates ribosomal RNA transcription. 920 86

The transcription factor TFIID is a multisubunit complex that is required for promoter recognition and accurate initiation of transcription by RNA polymerase II. To dissect the molecular architecture and the biochemical properties of TFIID, a small-scale density gradient sedimentation method is employed to separate related complexes through differences in their sedimentation properties. A small amount of starting material is sufficient to obtain readily assayable amounts of separated proteins after centrifugation for 8 to 12 h in a benchtop ultracentrifuge. Gradient fractions are analyzed by immunoblotting for the presence of specific components of TFIID. Sucrose gradient sedimentation is performed to separate a mixture of multiprotein complexes from a crude nuclear extract immunoprecipitation of the proteins present in each fraction with an anti-TBP antibody reveals multiple TBP-containing complexes of different sizes. Density gradient sedimentation permits separation of specific components in a complex mixture and preserves activity, allowing functional assays.
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PMID:Small-scale density gradient sedimentation to separate and analyze multiprotein complexes. 923 67

Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) is composed of three subunits: the TATA-binding protein, the TFIIB-related protein Brf, and B". TFIIIB, which is brought to RNA polymerase III-transcribed genes indirectly through interaction with DNA-bound TFIIIC or directly through DNA recognition by the TATA-binding protein, in turn recruits RNA polymerase III to the promoter. N-terminally deleted derivatives of Brf have been examined for their ability to interact with DNA-bound TFIIIC and with the other components of TFIIIB and for participation in transcription. Brf(165-596), lacking 164 N-proximal TFIIB-homologous amino acids, is competent to participate in the assembly of TFIIIB-DNA complexes and in TFIIIC-independent transcription. Even deletion of the entire TFIIB-homologous half of the protein, as in Brf(317-596) and Brf(352-596), allows some interaction with DNA-bound TBP and with the B" component of TFIIIB to be retained. The function of Brf(165-596) in transcription has also been examined in the context of B" with small internal deletions. The ability of Brf with this sizable N-terminal segment deleted to function in TFIIIC-independent transcription requires segments of B" that are individually indispensable although required on an either/or basis, in the context of complete Brf. These findings suggest a functional complementarity and reciprocity between the Brf and B" components of TFIIIB.
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PMID:Domains of the Brf component of RNA polymerase III transcription factor IIIB (TFIIIB): functions in assembly of TFIIIB-DNA complexes and recruitment of RNA polymerase to the promoter. 927 7

In eukaryotes, TFIIIB is required for proper initiation by RNA polymerase III. In the yeast Saccharomyces cerevisiae a single form of TFIIIB (gammaTFIIIB) is sufficient for transcription of all pol III genes, whereas in extracts derived from human cells two different hTFIIIB complexes exist which we have previously designated as hTFIIIB-alpha and hTFIIIB-beta. Human TFIIIB-alpha is a TBP-free entity and must be complemented by TBP for transcription of pol III genes driven by gene external promoters, whereas hTFIIIB-beta is a TBP-TAF complex which governs transcription from internal pol III promoters. We show that hTFIIIB-beta cannot be replaced by yeast TFIIIB for transcription of tRNA genes, but that the B" component of gammaTFIIIB can substitute for hTFIIIB-alpha activity in transcription of the human U6 gene. Moreover, hTFIIIB-alpha can be chromatographically divided into activities which are functionally related to gammaTFIIIE and recombinant yB"90, suggesting that hTFIIIB-alpha is a human homolog of yeast TFIIIB". In addition, we show that yeast TBP can only be exchanged against human TBP for in vitro transcription of the human and yeast U6 gene but virtually not for that of the yeast tRNA4Sup gene. This deficiency can be counteracted by a mutant of human TBP (R231K) which is able to replace yeast TBP for transcription of yeast tRNA genes in vitro.
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PMID:Functional interchangeability of TFIIIB components from yeast and human cells in vitro. 930 15

Affinity chromatography on columns containing the immobilized monomeric transcriptional elongation factor TFIIS or the essential large subunit, Elongin A, of the trimeric elongation factor, Elongin, was used to purify a human RNA polymerase II holoenzyme from HeLa whole cell extract. This holoenzyme contained nearstoichiometric amounts of all the general transcription factors, TFIIB, TFIID (TBP + TAFIIs), TFIIE, TFIIF, and TFIIH, required to accurately initiate transcription in vitro at the adenovirus major late promoter. It behaved as a large complex, slightly smaller than 70 S ribosomes, during gel filtration chromatography, and contained nearly half the TFIID that was present in the extract used for the affinity chromatography. It also contained the cyclin-dependent kinase CDK8, a human homologue of the Saccharomyces cerevisiae holoenzyme subunit SRB10, and many other polypeptides. Efficient interaction of holoenzyme with TFIIS or Elongin A required only the amino-terminal region of either protein. These regions are similar in amino acid sequence but dispensable for TFIIS or Elongin to regulate elongation in vitro by highly purified RNA polymerase II. The transcriptional activators GAL4-VP16 and GAL4-Sp1 activated transcription in vitro by purified holoenzyme in the absence of any additional factors.
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PMID:Interaction of elongation factors TFIIS and elongin A with a human RNA polymerase II holoenzyme capable of promoter-specific initiation and responsive to transcriptional activators. 930 22

The already complex process of transcription by RNA polymerase II has become even more complicated in the last few years with the identification of auxiliary factors in addition to the essential general initiation factors. In many cases these factors, which have been termed mediators or co-activators, are only required for activated or repressed transcription. In some cases the effects are specific for certain activators and repressors. Recently some of these auxiliary factors have been found in large complexes with either TBP, as TBP-associated factors (TAFs) in the general factor TFIID, or with pol II and a subset of the general factors, referred to as the 'holoenzyme'. Although the exact composition of these huge assemblies is still a matter of some debate, it is becoming clear that the complexes themselves come in more than one form. In particular, at least four forms of TFIID have been described, including one that contains a tissue-specific TAF and another with a cell type-specific form of TBP. In addition, in yeast there are at least two forms of the 'holoenzyme' distinguished by their mediator composition and by the spectrum of transcripts whose expression they affect. Genetic and biochemical analyses have begun to identify the interactions between the components of these complexes and the ever increasing family of DNA binding regulatory factors. These studies are complicated by the fact that individual regulatory factors often appear to have redundant interactions with multiple mediators. The existence of these different forms of transcription complexes defines a new target for regulation of subsets of eukaryotic genes.
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PMID:A multiplicity of mediators: alternative forms of transcription complexes communicate with transcriptional regulators. 939 88

Drosophila heat shock factor (HSF) binds to specific sequence elements of heat shock genes and can activate their transcription 200-fold. Though HSF has an acidic activation domain, the mechanistic details of heat shock gene activation remain undefined. Here we report that HSF interacts directly with the general transcription factor TBP (TATA-box binding protein), and these two factors bind cooperatively to heat shock promoters. A third factor that binds heat shock promoters, GAGA factor, also interacts with HSF and further stabilizes HSF binding to heat shock elements (HSEs). The interaction of HSF and TBP is explored in some detail here and is shown to be mediated by residues in both the amino- and carboxyl-terminal portions of HSF. This HSF/TBP interaction can be specifically disrupted by competition with the potent acidic transcriptional activator VP16. We further show that the acidic domain of the largest subunit of Drosophila RNA polymerase II (Pol II) associates with TBP in vitro and is specifically displaced from TBP upon addition of HSF. The region of TBP that mediates both HSF and Pol II acidic domain binding maps to the conserved carboxyl-terminal repeats and depends on at least one of the TBP residues known to be contacted by VP16 and to be critical for transcription activation. We discuss these findings in the context of a model in which HSF triggers hsp70 transcription by freeing the hsp70 promoter-paused Pol II from the constraints on elongation caused by the affinity of Pol II for general transcription factors.
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PMID:Cooperative and competitive protein interactions at the hsp70 promoter. 940 12

We have further analyzed the requirements of yeast RNA polymerase I (pol I) to initiate transcription at the ribosomal gene promoter. Resolution of yeast whole cell extracts through several chromatographic steps yielded three protein fractions required for accurate initiation. One fraction is composed of TBP associated within a 240 kDa protein complex. The fraction contributing the RNA polymerase I (pol I) activity consists of dimeric and monomeric pol I under conditions optimal for in vitro transcription. The capability to utilize the ribosomal gene promoter correlates with monomeric pol I complexes which are possibly associated with further transcription factors. These initiation competent pol I complexes appeared to be resistant to high salt concentrations. Pol I dimers which represent the majority of the isolated pol I, can be reversibly dissociated into monomers and are only active in non-specific RNA synthesis, if single stranded DNA serves as a template. We suggest a model in which dimeric inactive pol I is converted into an active monomeric form that might be associated with other transcription factors to maintain a stable initiation competent complex.
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PMID:Resolution of RNA polymerase I into dimers and monomers and their function in transcription. 946 42

PTF (PSE-binding transcription factor) activates transcription of snRNA and related genes. We investigated its distribution in HeLa nuclei by immunofluorescence, and found it spread throughout the nucleoplasm in small foci. In some cells, PTF is also concentrated in one, or very few, discrete regions (diameter approximately 1.3 micron) that appear during G1 phase and disappear in S phase. Oct1, a transcription factor that interacts with PTF, is also enriched in these domains; RNA polymerase II, TBP and Sp1 are also present. Each domain typically contains 2 or 3 transcription 'factories' where Br-UTP is incorporated into nascent transcripts. Accordingly, we have christened this region the Oct1/PTF/transcription (OPT) domain. It colocalizes with some, but not all, PIKA domains. It is distinct from other nuclear domains, including coiled bodies, gemini bodies, PML bodies and the perinucleolar compartment. A small region on chromosome 6 (band 6p21) containing only approximately 30 Mbp DNA, and chromosomes 6 and 7, associate with the domain significantly more than other chromosomes. The domains may act like nucleoli to bring particular genes on specific chromosomes together to a region where the appropriate transcription and processing factors are concentrated, thereby facilitating the expression of those genes.
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PMID:Regional and temporal specialization in the nucleus: a transcriptionally-active nuclear domain rich in PTF, Oct1 and PIKA antigens associates with specific chromosomes early in the cell cycle. 950 Oct 98


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