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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)

Basic mechanisms of transcription initiation are conserved from yeast to man. However, in contrast to genes transcribed by RNA polymerases II and III, ribosomal gene transcription by RNA polymerase I (Pol I) is species-specific. Promoter selectivity is mediated by SL1/TIF-IB, a multiprotein complex containing the TATA-binding protein (TBP) and TBP-associated factors (TAFs) which bind to DNA and nucleate the assembly of initiation complexes. Using a human cell line that expresses epitope-tagged yeast TBP, we have isolated a chimeric complex consisting of yeast TBP and human TAFs which faithfully promotes human rDNA transcription in vitro. This result argues that specific interactions between TBP and Pol I-specific TAFs have been evolutionarily conserved between distant species. In addition, this finding also underscores the importance of TAFs in determining promoter selectivity of Pol I.
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PMID:Yeast TBP can replace its human homologue in the RNA polymerase I-specific multisubunit factor SL1. 796 4

Unlike genes transcribed by RNA polymerases II and III, transcription by RNA polymerase I is highly species-specific. Ribosomal promoter selectivity is brought about by a multisubunit transcription factor (SL1/TIF-IB) which consists of the TATA-binding protein (TBP) and three TBP-associated factors (TAFs). To determine the basis for the inability of SL1/TIF-IB to recognize heterologous rDNA, the transcriptional properties and the subunit composition of the murine and the human factor, as well as a chimeric complex containing epitope-tagged human TBP and murine TAFs, have been compared. We show that TBP can be exchanged between the human and mouse factor indicating that the variable N-terminal domain of TBP does not play a significant role in rDNA promoter selectivity. Instead, DNA binding is brought about by the TAFs. UV crosslinking experiments demonstrate that binding to the ribosomal gene promoter is mediated by two TAFs (TAFI48 and TAFI68) which have the same electrophoretic mobility in the human and mouse factor. The largest TAF is different in both species and is suggested to play a role in the species-specific assembly of productive preinitiation complexes. Thus, evolutionary changes of rDNA promoter sequences have been accompanied by changes in specific TAFs.
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PMID:TBP-associated factors interact with DNA and govern species specificity of RNA polymerase I transcription. 801 60

We have characterized a transcription factor from Ehrlich ascites cells that is required for ribosomal gene transcription by RNA polymerase I (Pol I). This factor, termed TIF-IC, has a native molecular mass of 65 kDa, associates with Pol I, and is required both for the assembly of Sarkosyl-resistant initiation complexes and for the formation of the first internucleotide bonds. In addition to its function in transcription initiation, TIF-IC also plays a role in elongation of nascent RNA chains. At suboptimal levels of TIF-IC, transcripts with heterogeneous 3' ends are formed which are chased into full-length transcripts by the addition of more TIF-IC. Moreover, on a tailed template, which allows initiation in the absence of auxiliary factors, TIF-IC was found to stimulate the overall rate of transcription elongation and suppress pausing of Pol I. Thus TIF-IC appears to serve a function similar to the Pol II-specific factor TFIIF which is required for Pol II transcription initiation and elongation.
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PMID:TIF-IC, a factor involved in both transcription initiation and elongation of RNA polymerase I. 807 98

The 65-kDa herpes simplex virus type 1 encoded alpha trans-induction factor (alpha TIF or VP16) has two important functions: it is required for the efficient transcriptional induction of the alpha or immediate-early genes, and it acts as an essential structural component of the virion. The transcription properties of alpha TIF have been well studied in vitro. The protein is a powerful inducer of RNA polymerase II-directed transcription and, similar to the cellular transcriptional transactivators GAL4 and CGN4, contains separable DNA binding and transactivation domains. In contrast, little is known about the structural function of alpha TIF because this function can be studied only during virus replication and structural mutants are lethal. The in vivo analysis of alpha TIF is further complicated by the likelihood that the transcription and structural functions are not entirely separable. In this study, we take an alternate approach toward the development of alpha TIF mutants and their subsequent characterization. Rather than analyzing the effects of intragenic mutations, we have examined the properties of a mutant virus which expresses an alpha TIF fusion protein containing 61 amino acids of another herpes simplex virus type 1 virion protein, VP13/14, fused to its C terminus. This is the first report which demonstrates that the C-terminus of alpha TIF can tolerate the addition of an adjacent protein domain without compromising its transactivation function in vivo. Moreover, the VP13/14 sequences do not interfere with the protein-protein interactions required for virion targeting and assembly.
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PMID:An 85-kilodalton herpes simplex virus type 1 alpha trans-induction factor (VP16)-VP13/14 fusion protein retains the transactivation and structural properties of the wild-type molecule during virus infection. 810 36

The role of the Acanthamoeba castellanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor.
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PMID:TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors. 826 28

TIF-IB is a transcription factor which interacts with the mouse ribosomal gene promoter and nucleates the formation of an initiation complex containing RNA polymerase I (Pol I). We have purified this factor to near homogeneity and demonstrate that TIF-IB is a large complex (< 200 kDa) which contains several polypeptides. One of the subunits present in this protein complex is the TATA-binding protein (TBP) as revealed by copurification of TIF-IB activity and TBP over different chromatographic steps including immunoaffinity purification. In addition to TBP, three tightly associated proteins (TAFs-I) with apparent molecular weights of 95, 68, and 48 kDa are contained in this multimeric complex. This subunit composition is similar--but not identical--to the analogous human factor SL1. Depletion of TBP from TIF-IB-containing fractions by immunoprecipitation eliminates TIF-IB activity. Neither TBP alone nor fractions containing other TBP complexes are capable of substituting for TIF-IB activity. Therefore, TIF-IB is a unique complex with Pol I-specific TAFs distinct from other TBP-containing complexes. The identification of TBP as an integral part of the murine rDNA promoter-specific transcription initiation factor extends the previously noted similarity of transcriptional initiation by the three nuclear RNA polymerases and underscores the importance of TAFs in determining promoter specificity.
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PMID:A TBP-containing multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I. 841 71

Previously we have shown that the RNA polymerase I (Pol I)-specific transcription factor UBF stimulates transcription by both facilitating transcription complex formation and by relieving repression exerted by a negative-acting factor which competes for binding of the murine factor TIF-IB to the ribosomal gene promoter (1). We have purified and functionally characterized this repressor protein from Ehrlich ascites cells. The final preparation contained two polypeptides with molecular masses of 75 and 90 kDa, respectively. Both polypeptides interact with the rDNA promoter as revealed by UV-crosslinking experiments. The specificity of binding to the ribosomal gene promoter was demonstrated in an electrophoretic mobility shift assay and by DNase footprinting. The biochemical properties of this negative-acting factor closely resemble those of the Ku antigen, a human nuclear DNA-binding heterodimer which is the target of autoantibodies in several autoimmune diseases. Anti-Ku antibodies precipitate the repressor activity and overcome transcription inhibition. The data demonstrate that regulation of Pol I gene transcription may involve an antirepression mechanism as already documented for Pol II genes and suggest that Ku protein may be causally involved in repressor-mediated down regulation of rRNA synthesis.
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PMID:The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription. 850 46

A receptor tyrosine kinase (RTK), TIE (tyrosine kinase that contains immunoglobulin-like loops and epidermal growth factor [EGF] homology domains), is expressed in vascular endothelial and hematopoietic cells. We generated monoclonal antibodies (MoAbs) against the extracellular domain of TIE and a polyclonal antibody against the TIE carboxyterminus and used them to analyze expression of TIE in hematopoietic cells. Western blotting detected two forms of TIE protein with a molecular mass of 135 and 130 kD in hematopoietic and endothelial cells. Northern blotting analysis revealed that TIE was expressed preferentially in undifferentiated cell lines, especially when megakaryocytic, but not erythroid differentiation was induced. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that TIE was predominantly expressed in the human hematopoietic progenitor fraction, CD34+ cells. Fluorescence-activated cell sorting (FACS) showed that 42% of CD34+ and 17% of KIT-positive (KIT+) cells were TIE-positive (TIE+). The majority (81%) of the primitive hematopoietic stem cells, CD34+CD38- cells, were TIE+. Assays of progenitor cells and long-term culture-initiating cells (LTC-IC) showed that the TIE+ fraction contained more primitive cells than the TIE- fraction. Some TIE+ cells were in the CD34- fraction, which were CD19+ and CD20+ (B cells). These findings indicate that TIE has a unique spectrum of expression in primitive hematopoietic stem cells and B cells. Although its ligand has not been identified, TIE and its ligand may establish a novel regulatory pathway not only in early hematopoiesis, but also in the differentiation and/or proliferation of B cells.
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PMID:Predominant expression of a receptor tyrosine kinase, TIE, in hematopoietic stem cells and B cells. 854 81

Tumour-secreted vascular endothelial growth factor (VEGF) exerts a number of effects which are important in tumour pathology, including stimulation of angiogenesis and permeabilisation of tumour-associated vasculature. In this study we have examined the possibility that VEGF may also play an autocrine role in tumour growth. Using reverse-transcriptase polymerase chain reaction (RT-PCR), the expression of VEGF was found in 15/15 human tumour cell lines examined, while the VEGF receptor KDR was detected only in three melanoma cell lines (MeWo and A375, both wild type and metastatic variant). Exogenously added VEGF (10ng/ml) was able to stimulate up to 40% increased proliferation of A375 M melanoma cells following a 48-h period of quiescence, suggesting that VEGF may indeed play a role in autocrine, as well as paracrine, stimulation of melanoma growth.
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PMID:Melanoma cell lines express VEGF receptor KDR and respond to exogenously added VEGF. 855 90

The cyclin-dependent kinase (CDK)-activating kinase, CAK, from mammals and amphibians consists of MO15/CDK7 and cyclin H, a complex which has been identified also as a RNA polymerase II C-terminal domain (CTD) kinase. While the Schizosaccharomyces pombe cdc2 gene product also requires an activating phosphorylation, the enzyme responsible has not been identified. We have isolated an essential S.pombe gene, mop1, whose product is closely related to MO15 and to Saccharomyces cerevisiae Kin28. The functional similarity of Mop1 and MO15 is reflected in the ability of MO15 to rescue a mop1 null allele. This suggests that Mop1 would be a CDK, and indeed Mop1 associates with a previously characterized cyclin H-related cyclin Mcs2 of S.pombe. Also, Mop1 and Mcs2 can associate with the heterologous partners human cyclin H and MO15, respectively. Moreover, the rescue of a temperature-sensitive mcs2 strain by expression of mop1+ demonstrates a genetic interaction between mop1 and mcs2. In a functional assay, immunoprecipitated Mop1-Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. The CAK activity of Mop1-Mcs2 distinguishes it from the related CDK-cyclin pair Kin28-Ccl1 from S.cerevisiae, and supports the notion that Mop1-Mcs2 may represent a homolog of MO15-cyclin H in S.pombe with apparent dual roles as a RNA polymerase CTD kinase and as a CAK.
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PMID:Schizosaccharomyces pombe Mop1-Mcs2 is related to mammalian CAK. 855 36


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