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 global anaerobic regulator FNR is a DNA binding protein that activates transcription of genes required for anaerobic metabolism in Escherichia coli through interactions with RNA polymerase (RNAP). Alanine-scanning mutagenesis of FNR amino acid residues 181 to 193 of FNR was utilized to determine which amino acid side chains are required for transcription of both class II and class I promoters. In vivo assays of FNR function demonstrated that a core of residues (F181, R184, S187, and R189) was required for efficient activation of class II promoters, while at a class I promoter, FF(-61.5), only S187 and R189 were critical for FNR activation. Site-directed mutagenesis of positions 184, 187, and 189 revealed that the positive charge contributes to the function of the side chain at positions 184 and 189 while the serine hydroxyl is critical for the function of position 187. Subsequent analysis of the carboxy-terminal domain of the alpha subunit (alphaCTD) of RNAP, using an alanine library in single copy, revealed that in addition to previously characterized side chains (D305, R317, and L318), E286 and E288 contributed to FNR activation of both class II and class I promoters, suggesting that alphaCTD region 285 to 288 also participates in activation by FNR. In conclusion, this study demonstrates that multiple side chains within region 181 to 192 are required for FNR activation and the surface of alphaCTD required for FNR activation is more extensive than previously observed.
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PMID:Additional determinants within Escherichia coli FNR activating region 1 and RNA polymerase alpha subunit required for transcription activation. 1571 44

Transcriptional elongation of most eukaryotic genes by RNA polymerase II requires the kinase activity of the positive transcription elongation factor b (P-TEFb). The catalytically active P-TEFb complex becomes inactive when sequestered into the large complex by the cooperative actions of 7SK snRNA and HEXIM1. In this study, we report that HEXIM1 forms oligomers in cells. This oligomerization is mediated by its predicted coiled-coil region in the C-terminal domain and 7SK snRNA that binds a basic region within the central part of HEXIM1. Alanine-mutagenesis of evolutionary conserved leucines in the coiled-coil region and the digestion of 7SK snRNA by RNase A treatment prevent this oligomerization. Importantly, mutations of the N-terminal part of the coiled-coil region abrogate the ability of HEXIM1 to bind and inhibit P-TEFb. Finally, the formation of HEXIM1 oligomers via the C-terminal part of the coiled-coil or basic regions is critical for the inhibition of transcription. Our results suggest that two independent regions in HEXIM1 form oligomers to incorporate P-TEFb into the large complex and determine the inhibition of transcriptional elongation.
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PMID:Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb. 1637 79

Panicum mosaic virus (PMV) has a positive-sense, single-stranded RNA genome that serves as the mRNA for two 5'-proximal genes, p48 and p112. The p112 open reading frame (ORF) has a GDD-motif, a feature of virus RNA-dependent RNA polymerases. Replication assays in protoplasts showed that p48 and p112 are sufficient for replication of PMV and its satellite virus (SPMV). Differential centrifugation of extracts from PMV-infected plants showed that the p48 and p112 proteins are membrane-associated. The same fractions exhibited RNA polymerase activity in vitro on viral RNA templates, suggesting that p48 and p112 represent the viral replication proteins. Moreover, we identified a domain spanning amino acids 306 to 405 on the p48 and p112 PMV ORFs that is common to the Tombusviridae. Alanine scanning mutagenesis of the conserved domain (CD) revealed that several substitutions were lethal or severely debilitated PMV accumulation. Other substitutions did not affect RNA accumulation, yet they caused variable phenotypes suggestive of plant-dependent effects on systemic invasion and symptom induction. The mutants that were most debilitating to PMV replication were hydrophobic amino acids that we hypothesize are important for membrane localization and functional replicase activity.
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PMID:Panicovirus accumulation is governed by two membrane-associated proteins with a newly identified conserved motif that contributes to pathogenicity. 1652 73

The Spx protein of Bacillus subtilis exerts both positive and negative transcriptional control in response to oxidative stress by interacting with the C-terminal domain of the RNA polymerase (RNAP) alpha subunit (alphaCTD). Thus, transcription of the srf operon at the onset of competence development, which requires the ComA response regulator of the ComPA signal transduction system, is repressed by Spx-alphaCTD interaction. Previous genetic and structural analyses have determined that an Spx-binding surface resides in and around the alpha1 region of alphaCTD. Alanine-scanning mutagenesis of B. subtilis alphaCTD uncovered residue positions required for Spx function and ComA-dependent srf transcriptional activation. Analysis of srf-lacZ fusion expression, DNase I footprinting, and solid-phase promoter retention experiments indicate that Spx interferes with ComA-alphaCTD interaction and that residues Y263, C265, and K267 of the alpha1 region lie within overlapping ComA- and Spx-binding sites for alphaCTD interaction. Evidence is also presented that oxidized Spx, while enhancing interference of activator-RNAP interaction, is not essential for negative control.
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PMID:Mutational analysis of the Bacillus subtilis RNA polymerase alpha C-terminal domain supports the interference model of Spx-dependent repression. 1674 Sep 36

TATA-binding protein-associated factor 1 (TAF1) is an essential component of the general transcription factor IID (TFIID), which nucleates assembly of the preinitiation complex for transcription by RNA polymerase II. TATA-binding protein and TAF1.TAF2 heterodimers are the only components of TFIID shown to bind specific DNA sequences (the TATA box and initiator, respectively), raising the question of how TFIID localizes to gene promoters that lack binding sites for these proteins. Here we demonstrate that Drosophila TAF1 protein isoforms TAF1-2 and TAF1-4 directly bind DNA independently of TAF2. DNA binding by TAF1 isoforms is mediated by cooperative interactions of two identical AT-hook motifs, one of which is encoded by an alternatively spliced exon. Electrophoretic mobility shift assays revealed that TAF1-2 bound the minor groove of adenine-thymine-rich DNA with a preference for the sequence AAT. Alanine-scanning mutagenesis of the alternatively spliced AT-hook indicated that Lys and Arg residues made essential DNA contacts, whereas Gly and Pro residues within the Arg-Gly-Arg-Pro core sequence were less important for DNA binding, suggesting that AT-hooks are more divergent than previously predicted. TAF1-2 bound with variable affinity to the transcription start site of several Drosophila genes, and binding to the hsp70 promoter was reduced by mutation of a single base pair at the transcription start site. Collectively, these data indicate that AT-hooks serve to anchor TAF1 isoforms to the minor groove of adenine-thymine-rich Drosophila gene promoters and suggest a model in which regulated expression of TAF1 isoforms by alternative splicing contributes to gene-specific transcription.
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PMID:DNA binding properties of TAF1 isoforms with two AT-hooks. 1689 81

The binding of transcription factor (TF) IIIA to the internal control region of the 5 S RNA gene is the first step in the assembly of a DNA-TFIIIA-TFIIIC- TFIIIB transcription complex, which promotes accurate transcription by RNA polymerase III. With the use of mutations that are predicted to disrupt the folding of a zinc finger, we have examined the roles of zinc fingers 1 through 7 of yeast TFIIIA in the establishment of a functional transcription complex both in vitro and in vivo. Our data indicate that, in addition to their role in DNA binding, the first and seventh zinc fingers contribute other essential roles in the assembly of an active transcription complex. Alanine-scanning mutagenesis identified residues within zinc finger 1 that are not required for DNA binding but are required for incorporation of TFIIIC into the TFIIIA-DNA complex. Although disruption of zinc finger 2 or 3 had a deleterious effect on the activity of TFIIIA both in vitro and in vivo, we found that increasing the level of their in vivo expression allowed these mutant proteins to support cell viability. Disruption of zinc fingers 4, 5 or 6 had minimal effect on the DNA binding and TF activities of TFIIIA.
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PMID:Zinc fingers 1 and 7 of yeast TFIIIA are essential for assembly of a functional transcription complex on the 5 S RNA gene. 1762 45

Heterochromatin formation involves the nucleation and spreading of structural and epigenetic features along the chromatin fiber. Chromatin barriers and associated proteins counteract the spreading of heterochromatin, thereby restricting it to specific regions of the genome. We have performed gene expression studies and chromatin immunoprecipitation on strains in which native centromere sequences have been mutated to study the mechanism by which a tRNA(Alanine) gene barrier (cen1 tDNA(Ala)) blocks the spread of pericentromeric heterochromatin at the centromere of chromosome 1 (cen1) in the fission yeast, Schizosaccharomyces pombe. Within the centromere, barrier activity is a general property of tDNAs and, unlike previously characterized barriers, requires the association of both transcription factor IIIC and RNA Polymerase III. Although the cen1 tDNA(Ala) gene is actively transcribed, barrier activity is independent of transcriptional orientation. These findings provide experimental evidence for the involvement of a fully assembled RNA polymerase III transcription complex in defining independent structural and functional domains at a eukaryotic centromere.
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PMID:An RNA polymerase III-dependent heterochromatin barrier at fission yeast centromere 1. 1797 62

The XylS protein is the positive transcription regulator of the TOL plasmid meta-cleavage pathway operon Pm. XylS belongs to the AraC family of transcriptional regulators and exhibits an N-terminal domain involved in effector recognition, and a C-terminal domain, made up of seven alpha-helices conforming two helix-turn-helix DNA-binding domains. alpha-Helix 3 and alpha-helix 6 are the recognition helices. In consonance with XylS structural organization, Pm exhibits a bipartite DNA-binding motif consisting of two boxes, called A and B, whose sequences are TGCA and GGNTA, respectively. This bipartite motif is repeated at the Pm promoter so that one of the XylS monomers binds to each of the repeats. An extensive series of genetic epistasis assays combining mutant Pm promoters and XylS single substitution mutant proteins revealed that alpha-helix 3 contacts A box nucleotides, whereas alpha-helix 6 residues contact B box nucleotides. In alpha-helix 3, Asn246 and Arg242 are involved in specific contacts with the TG dinucleotide at box A, whereas Arg296 and Glu299 contact the second G and T nucleotides at box B. On the basis of our results and of the three-dimensional model of the XylS C-terminal domain, we propose that Ser243, Glu249 and Lys250 in alpha-helix 3, and Asn299 and Arg302 in alpha-helix 6 contact the phosphate backbones. Alanine substitutions at the predicted phosphate backbone-contacting residues yielded mutants with low levels of activity, suggesting that XylS-Pm binding specificity not only involves specific amino acid-base interactions, but also relies on secondary DNA structure, which, although at another level, also comes into play. We propose a model in which a XylS dimer binds to the direct repeats in Pm in a head-to-tail conformation that allows the direct interaction of the XylS proximal subunit with the RNA polymerase sigma factor.
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PMID:XylS-Pm promoter interactions through two helix-turn-helix motifs: identifying XylS residues important for DNA binding and activation. 1800 85

Hepatitis C virus infection constitutes a serious health problem in need of more effective therapies. Nucleoside analogues with improved exposure, efficacy, and selectivity are recognized as likely key components of future HCV therapy. 2'-C-Methylguanosine triphosphate has been known as a potent inhibitor of HCV RNA polymerase for some time, but the parent nucleoside is only moderately active due to poor intracellular phosphorylation. We herein report the application of phosphoramidate ProTide technology to bypass the rate-limiting initial phosphorylation of this nucleoside. Over 30 novel ProTides are reported, with variations in the aryl, ester, and amino acid regions. l-Alanine compounds are recognized as potent and selective inhibitors of HCV in replicon assay but lack rodent plasma stability despite considerable ester variation. Amino acid variation retaining the lead benzyl ester moiety gives an increase in rodent stability but at the cost of potency. Finally l-valine esters with ester variation lead to potent, stable compounds. Pharmacokinetic studies on these agents in the mouse reveal liver exposure to the bioactive triphosphate species following single oral dosing. Systemic exposure of the ProTide and parent nucleoside are low, indicating possible low toxicity in vivo, while liver concentrations of the active species may be predictive of efficacy in the clinic. This represents one of the most thorough cross-species studies of ProTides to date.
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PMID:Phosphoramidate ProTides of 2'-C-methylguanosine as highly potent inhibitors of hepatitis C virus. Study of their in vitro and in vivo properties. 2052 90

The AsiA protein is a T4 bacteriophage early gene product that regulates transcription of host and viral genes. Monomeric AsiA binds tightly to the sigma(70) subunit of Escherichia coli RNA polymerase, thereby inhibiting transcription from bacterial promoters and phage early promoters and coactivating transcription from phage middle promoters. Results of structural studies have identified amino acids at the protomer-protomer interface in dimeric AsiA and at the monomeric AsiA-sigma(70) interface and demonstrated substantial overlap in the sets of residues that comprise each. Here we evaluate the contributions of individual interfacial amino acid side chains to protomer-protomer affinity in AsiA homodimers, to monomeric AsiA affinity for sigma(70), and to AsiA function in transcription. Sedimentation equilibrium, dynamic light scattering, electrophoretic mobility shift, and transcription activity measurements were used to assess affinity and function of site-specific AsiA mutants. Alanine substitutions for solvent-inaccessible residues positioned centrally in the protomer-protomer interface of the AsiA homodimer, V14, I17, and I40, resulted in the largest changes in free energy of dimer association, whereas alanine substitutions at other interfacial positions had little effect. These residues also contribute significantly to AsiA-dependent regulation of RNA polymerase activity, as do additional residues positioned at the periphery of the interface (K20 and F21). Notably, the relative contributions of a given amino acid side chain to RNA polymerase inhibition and activation (MotA-independent) by AsiA are very similar in most cases. The mainstay for intermolecular affinity and AsiA function appears to be I17. Our results define the core interfacial residues of AsiA, establish roles for many of the interfacial amino acids, are in agreement with the tenets underlying protein-protein interactions and interfaces, and will be beneficial for a general, comprehensive understanding of the mechanistic underpinnings of bacterial RNA polymerase regulation.
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PMID:Determinants of affinity and activity of the anti-sigma factor AsiA. 2054 5


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