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)

Transcription factor IIIB (TFIIIB), consisting of the TATA-binding protein (TBP), TFIIB-related factor (Brf1) and Bdp1, is a central component in basal and regulated transcription by RNA polymerase III. TFIIIB recruits its polymerase to the promoter and subsequently has an essential role in the formation of the open initiation complex. The amino-terminal half of Brf1 shares a high degree of sequence similarity with the polymerase II general transcription factor TFIIB, but it is the carboxy-terminal half of Brf1 that contributes most of its binding affinity with TBP. The principal anchoring region is located between residues 435 and 545 of yeast Brf1, comprising its homology domain II. The same region also provides the primary interface for assembling Bdp1 into the TFIIIB complex. We report here a 2.95 A resolution crystal structure of the ternary complex containing Brf1 homology domain II, the conserved region of TBP and 19 base pairs of U6 promoter DNA. The structure reveals the core interface for assembly of TFIIIB and demonstrates how the loosely packed Brf1 domain achieves remarkable binding specificity with the convex and lateral surfaces of TBP.
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PMID:Crystal structure of a transcription factor IIIB core interface ternary complex. 1266 Jul 36

RNA polymerase II-dependent transcription requires the assembly of a multi-protein, preinitiation complex on core promoter elements. Transcription factor IID (TFIID) comprising the TATA box-binding protein (TBP) and TBP-associated factors (TAFs) is responsible for promoter recognition in this complex. Subsequent association of TFIIA and TFIIB provides enhanced complex stability. TFIIA is required for transcriptional stimulation by certain viral and cellular activators, and favors formation of the preinitiation complex in the presence of repressor NC2. The X-ray structures of human and yeast TBP/TFIIA/DNA complexes at 2.1A and 1.9A resolution, respectively, are presented here and seen to resemble each other closely. The interactions made by human TFIIA with TBP and DNA within and upstream of the TATA box, including those involving water molecules, are described and compared to the yeast structure. Of particular interest is a previously unobserved region of TFIIA that extends the binding interface with TBP in the yeast, but not in the human complex, and that further elucidates biochemical and genetic results.
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PMID:Novel interactions between the components of human and yeast TFIIA/TBP/DNA complexes. 1297 51

Transcription factor IIE (TFIIE) is a general initiation and promoter escape factor for RNA polymerase II composed of p56 (TFIIE-alpha) and p34 (TFIIE-beta) subunits. Our laboratories experienced difficulty producing adequate quantities of recombinant human TFIIE-alpha for in vitro studies using available clones. We therefore re-engineered the TFIIE subunit production vectors and tested various Escherichia coli host strains to optimize expression. We report a much-improved system for production of pure, soluble, and active TFIIE complex for in vitro studies.
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PMID:Efficient production of recombinant human transcription factor IIE. 1500 67

Transcription factor IIF (TFIIF) is required for transcription of protein-encoding genes by eukaryotic RNA polymerase II. In contrast to numerous studies establishing a role for higher eukaryotic TFIIF in multiple steps of the transcription cycle, relatively little has been reported regarding the functions of TFIIF in the yeast Saccharomyces cerevisiae. In this study, site-directed mutagenesis, plasmid shuffle complementation assays, and primer extension analyses were employed to probe the functional domains of the S. cerevisiae TFIIF subunits Tfg1 and Tfg2. Analyses of 35 Tfg1 alanine substitution mutants and 19 Tfg2 substitution mutants identified 5 mutants exhibiting altered properties in vivo. Primer extension analyses revealed that the conditional growth properties exhibited by the tfg1-E346A, tfg1-W350A, and tfg2-L59K mutants were associated with pronounced upstream shifts in transcription initiation in vivo. Analyses of double mutant strains demonstrated functional interactions between the Tfg1 mutations and mutations in Tfg2, TFIIB, and RNA polymerase II. Importantly, biochemical results demonstrated an altered interaction between mutant TFIIF protein and RNA polymerase II. These results provide direct evidence for the involvement of S. cerevisiae TFIIF in the mechanism of transcription start site utilization and support the view that a TFIIF-RNA polymerase II interaction is a determinant in this process.
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PMID:Amino acid substitutions in yeast TFIIF confer upstream shifts in transcription initiation and altered interaction with RNA polymerase II. 1557 98

Transcription factor (TF) IIIB, the central transcription initiation factor of RNA polymerase III (pol III), is composed of three subunits, Bdp1, Brf1 and TATA-binding protein (TBP), all essential for normal function in vivo and in vitro. Brf1 is a modular protein: Its N-proximal half is related to TFIIB and binds similarly to the C-terminal stirrup of TBP; its C-proximal one-third provides most of the affinity for TBP by binding along the entire length of the convex surface and N-terminal lateral face of TBP. A structure-informed triple fusion protein, with TBP core placed between the N- and C-proximal domains of Brf1, has been constructed. The Brf1-TBP triple fusion protein effectively replaces both Brf1 and TBP in TFIIIC-dependent and -independent transcription in vitro, and forms extremely stable TFIIIB-DNA complexes that are indistinguishable from wild-type TFIIIB-DNA complexes by chemical nuclease footprinting. Unlike Brf1 and TBP, the triple fusion protein is able to recruit pol III for TATA box-directed transcription of linear and supercoiled DNA in the absence of Bdp1. The Brf1-TBP triple fusion protein also effectively replaces Brf1 function in vivo as the intact protein, creating a TBP paralogue in yeast that is privatized for pol III transcription.
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PMID:Reconfiguring the connectivity of a multiprotein complex: fusions of yeast TATA-binding protein with Brf1, and the function of transcription factor IIIB. 1622 32

Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.
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PMID:Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors. 1651 97

Transcription factor IIA (TFIIA) is one of the general transcription factors for RNA polymerase II and composed of three subunits, TFIIAalpha, TFIIAbeta and TFIIAgamma. TFIIAalpha and TFIIAbeta are encoded by a single gene (TFIIAalphabeta) and mature through internal cleavage of TFIIAalphabeta. In this study, we found that structures of TFIIAalphabeta and TFIIAgamma are highly homologous with each mammalian counterpart. Exon-intron organizations of the human and chicken TFIIA genes were also homologous. The sequence of the cleavage region of the chicken TFIIAalphabeta precursor protein was fitted to the consensus cleavage recognition site. It was thus demonstrated that TFIIA is conserved in vertebrates. TFIIA proteins are present ubiquitously in chicken tissues. Fluorescent in situ hybridization revealed that TFIIAalphabeta and TFIIAgamma genes are located in chromosome 5 and a mini-chromosome, respectively. We generated semi-knockout chicken DT40 cells for TFIIAalphabeta and TFIIAgamma genes with high homologous recombination efficiencies, whereas we failed to establish double-knockout cells for each gene. It is thought that both genes for TFIIA are required in vertebrates. TFIIA siRNA resulted in deceleration of cell growth rate, suggesting that, consistent with those of knockout assays, TFIIA is associated with cell growth regulation.
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PMID:Chromosomal position, structure, expression, and requirement of genes for chicken transcription factor IIA. 1754 29

We determined the effect of the N-terminal histone tails on nucleosome traversal by yeast and human RNA polymerase II (pol II). Removal of H2A/H2B tails, H3/H4 tails, or all tails increased complete traversal of the nucleosome by human pol II, although the increase varied considerably depending on the template and on which tails were removed. Human pol II achieved >80% traversal of one nucleosomal template lacking the H2A/H2B tails, but even in those reactions, the transcript elongation rate was lower than the rate on pure DNA templates. For yeast pol II, transcription proceeded much farther into the nucleosome in the absence of tails, but complete read-through was not substantially increased by tail removal. Transcription factor IIS provided roughly the same level of read-through stimulation for transcript elongation in the presence or absence of tails. FACT also stimulated elongation on nucleosomal templates, and this effect was similar regardless of the presence of tails. For both polymerases, removal of the H2A/H2B tails reduced pausing throughout the nucleosome, suggesting that histone tails affect a common step at most points during nucleosome traversal. We conclude that histone tails provide a significant part of the nucleosomal barrier to pol II transcript elongation.
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PMID:Histone N-terminal tails interfere with nucleosome traversal by RNA polymerase II. 1881 26

Transcription factor sigma(28) in Chlamydia trachomatis (sigma(28)(Ct)) plays a role in the regulation of genes that are important for late-stage morphological differentiation. In vitro mutational and genetic screening in Salmonella enterica serovar Typhimurium was performed in order to identify mutants with mutations in region 4 of sigma(28)(Ct) that were defective in sigma(28)-specific transcription. Specially, the previously undefined but important interactions between sigma(28)(Ct) region 4 and the flap domain of the RNA polymerase beta subunit (beta-flap) or the -35 element of the chlamydial hctB promoter were examined. Our results indicate that amino acid residues E206, Y214, and E222 of sigma(28)(Ct) contribute to an interaction with the beta-flap when sigma(28)(Ct) associates with the core RNA polymerase. These residues function in contacts with the beta-flap similarly to their counterpart residues in Escherichia coli sigma(70). Conversely, residue Q236 of sigma(28)(Ct) directly binds the chlamydial hctB -35 element. The conserved counterpart residue in E. coli sigma(70) has not been reported to interact with the -35 element of the sigma(70) promoter. Observed functional disparity between sigma(28)(Ct) and sigma(70) region 4 is consistent with their divergent properties in promoter recognition. This work provides new insight into understanding the molecular basis of gene regulation controlled by sigma(28)(Ct) in C. trachomatis.
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PMID:Mutagenesis of region 4 of sigma 28 from Chlamydia trachomatis defines determinants for protein-protein and protein-DNA interactions. 1897 51

Transcription factor TFIIIB plays key roles in transcription by RNA polymerase III. Its three components (TBP, Brf1, and Bdp1) participate in crucial molecular events that include RNA polymerase recruitment, formation of the open initiation complex, and recycling of transcription. Although the details of the interaction among DNA, TBP, and Brf1 have been, in part, revealed through the crystal structure of their ternary complex, structural details of the Brf1-Bdp1 interaction are lacking. In this paper, nuclear magnetic resonance (NMR) is used to map the interaction interface between Bdp1 and Brf1 at single-amino acid resolution, using minimal functional segments of the two proteins. An NMR-derived structural model shows that the principal anchorage site of Brf1 is located on a convex surface of Bdp1 that encompasses helix 1 and helix 3 of its conserved SANT domain. The main Bdp1 anchorage site is provided by a small set of residues belonging to a Brf1 segment of residues 470-495.
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PMID:Structural characterization of the interaction between TFIIIB components Bdp1 and Brf1. 1908 69


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