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)

Fundamental questions in bacterial gene regulation concern how multiple regulatory proteins interact with the transcription apparatus at a single promoter and what are the roles of protein contacts with RNA polymerase and changes in DNA conformation. Transcription of the Escherichia coli uhpT gene, encoding the inducible sugar phosphate transporter, is dependent on the response regulator UhpA and is stimulated by the cyclic AMP receptor protein (CAP). UhpA binds to multiple sites in the uhpT promoter between positions -80 and -32 upstream of the transcription start site, and CAP binds to a single site centered at position -103.5. The role in uhpT transcription of portions of RNA polymerase Esigma(70) holoenzyme which affect regulation at other promoters was examined by using series of alanine substitutions throughout the C-terminal domains of RpoA (residues 255 to 329) and of RpoD (residues 570 to 613). Alanine substitutions that affected in vivo expression of a uhpT-lacZ transcriptional fusion were tested for their effect on in vitro transcription activity by using reconstituted holoenzymes. Consistent with the binding of UhpA near the -35 region, residues K593 and K599 in the C-terminal region of RpoD were necessary for efficient uhpT expression in response to UhpA alone. Their requirement was overcome when CAP was also present. In addition, residues R265, G296, and S299 in the DNA-binding surface of the C-terminal domain of RpoA (alphaCTD) were important for uhpT transcription even in the presence of CAP. Substitutions at several other positions had effects in cells but not during in vitro transcription with saturating levels of the transcription factors. Two DNase-hypersensitive sites near the upstream end of the UhpA-binding region were seen in the presence of all three transcription factors. Their appearance required functional alphaCTD but not the presence of upstream DNA. These results suggest that both transcription activators depend on or interact with different subunits of RNA polymerase, although their role in formation of proper DNA geometry may also be crucial.
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PMID:RNA polymerase alpha and sigma(70) subunits participate in transcription of the Escherichia coli uhpT promoter. 1057 30

Virus-encoded mRNA capping enzymes are attractive targets for antiviral therapy, but functional studies have been limited by the lack of genetically tractable in vivo systems that focus exclusively on the RNA-processing activities of the viral proteins. Here we have developed such a system by engineering a viral capping enzyme-vaccinia virus D1(1-545)p, an RNA triphosphatase and RNA guanylyltransferase-to function in the budding yeast Saccharomyces cerevisiae in lieu of the endogenous fungal triphosphatase (Cet1p) and guanylyltransferase (Ceg1p). This was accomplished by fusion of D1(1-545)p to the C-terminal guanylyltransferase domain of mammalian capping enzyme, Mce1(211-597)p, which serves as a vehicle to target the viral capping enzyme to the RNA polymerase II elongation complex. An inactivating mutation (K294A) of the mammalian guanylyltransferase active site in the fusion protein had no impact on genetic complementation of cet1Deltaceg1Delta cells, thus proving that (i) the viral guanylyltransferase was active in vivo and (ii) the mammalian domain can serve purely as a chaperone to direct other proteins to the transcription complex. Alanine scanning had identified five amino acids of vaccinia virus capping enzyme-Glu37, Glu39, Arg77, Glu192, and Glu194-that are essential for gamma phosphate cleavage in vitro. Here we show that the introduction of mutation E37A, R77A, or E192A into the fusion protein abrogates RNA triphosphatase function in vivo. The essential residues are located within three motifs that define a family of viral and fungal metal-dependent phosphohydrolases with a distinctive capacity to hydrolyze nucleoside triphosphates to nucleoside diphosphates in the presence of manganese or cobalt. The acidic residues Glu37, Glu39, and Glu192 likely comprise the metal-binding site of vaccinia virus triphosphatase, insofar as their replacement by glutamine abolishes the RNA triphosphatase and ATPase activities.
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PMID:A yeast-based genetic system for functional analysis of viral mRNA capping enzymes. 1082 53

Deletions within the thumb subdomain (residues 335-408) of T7 RNA polymerase decrease elongation complex stability and processivity, but the structure of a T7RNAP initial transcription complex containing a 3-nucleotide RNA reveals no interactions between the thumb and the RNA or DNA. Modeling of a longer RNA in this structure, using a T7DNAP-primer-template structure as a guide, suggests that the phosphate ribose backbone of the RNA contacts a stretch of mostly positively charged side chains between residues 385 and 395 of helix N of the thumb. Scanning mutagenesis of this region reveals that alanine substitutions of Arg(391), Ser(393), and Arg(394) destabilize elongation complexes and that substitutions at 393 and 394 increase termination of transcripts 5 or more bases in length. The alpha-carbons of all 3 of these residues lie on the side of helix N, which faces into the template-binding cleft of the RNA polymerase, and modeling suggests that they can contact the RNA 4-5 bases away from the 3'-end. Alanine substitutions of other residues within 385-395 do not have marked effects on transcription complex stability, but alanine substitutions of Asp(388) and Tyr(385) reduce pausing and termination at the T7 concatemer junction. Both of these side chains lie on the outer side of helix N, pointing away from the template binding cleft. The thumb subdomain of T7RNAP therefore has roles both in transcription complex stabilization and in pausing and termination at the T7 concatemer junction.
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PMID:Scanning mutagenesis reveals roles for helix n of the bacteriophage T7 RNA polymerase thumb subdomain in transcription complex stability, pausing, and termination. 1112 63

The transcriptional activator CooA from Rhodospirillum rubrum contains a six-coordinate protoheme that acts as a CO sensor in vivo. CO is a physiological effector of CooA and replaces one of the axial ligands of the ferrous heme to form the CO-bound CooA that is active as the transcriptional activator. Cys75 or His77 is coordinated to the ferric and ferrous hemes in CooA, respectively. The redox-controlled ligand exchange between Cys75 and His77 proceeds during the change in the redox state of the heme. The reduction and oxidation midpoint potentials of CooA have been determined to be -320 and -260 mV, respectively. The properties of a functional chimera derived from CRP and CooA suggest that CooA activates the transcription by a similar mechanism to that for CRP at Class II CRP-dependent promoters. Alanine-scanning mutagenesis has revealed that Arg24 and Arg53 of CooA, which will be concerned with the protein-protein interaction with RNA polymerase, are critical amino acid residues for the transcriptional activator activity of CooA, and that Lys26 and Asp94 modulate the activity of CooA.
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PMID:CO sensing and regulation of gene expression by the transcriptional activator CooA. 1113 38

Alanine scanning of the Escherichia coli RNA polymerase alpha subunit C-terminal domain (alphaCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of alphaCTD affected class I CRP-dependent transcription from the CC(-61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for alphaCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in alphaCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.
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PMID:Determinants of the C-terminal domain of the Escherichia coli RNA polymerase alpha subunit important for transcription at class I cyclic AMP receptor protein-dependent promoters. 1191 59

SoxS is the direct transcriptional activator of the superoxide regulon. SoxS recognizes a highly degenerate "soxbox" DNA sequence, and activates transcription from class I and class II promoters. SoxS is the smallest member of the AraC/XylS family of transcription regulators whose hallmark is dual helix-turn-helix (HTH) DNA-binding motifs. Evidence suggests that the N-terminal HTH motif of SoxS interacts with a highly conserved region of the soxbox termed recognition element 1 (RE1), while the C-terminal HTH motif interacts with the less conserved recognition element 2 (RE2). In the work described here, we prepared a complete library of 101 SoxS mutants containing single alanine substitutions of SoxS, and we characterized the mutant proteins in vivo and in vitro. With SoxS being closely related to MarA, we analyzed the effects of the SoxS mutations in the context of the MarA-mar crystal structure and with respect to the NMR study of MarA-DNA complexes in solution. From the properties of the alanine substitutions, we conclude the following. (1) Surface-exposed residues of helix 3 and helix 6, the recognition helices of the dual HTH motifs, are important to DNA binding and transcription activation; however, substitutions of residues predicted from the MarA-mar crystal structure to make contact with the sugar-phosphate backbone are more detrimental to DNA binding than mutations predicted to make base-specific contacts. (2) Substitution of several residues within the recognition helix predicted to make base-specific contacts with RE2 have relatively little effect on DNA-binding, suggesting the possibility of alternative protein-DNA interactions than those inferred from the MarA-mar crystal structure. (3) DNA binding and transcription activation were reduced by substitution of conserved amino acid residues comprising the hydrophobic core, presumably because they disrupt the structural integrity of SoxS. (4) Mutant K30A appears to be a positive control mutant defective in a protein-protein interaction with RNA polymerase that is required for transcription activation at all SoxS-dependent promoters because it binds and bends DNA normally but fails to activate transcription from both classes of promoters. Alanine substitutions of surface-exposed residues H3, K5, D9, S31, and V45 confer a similar phenotype. Since these residues are near K30 on the surface of the protein, the surface formed by the six residues may be used to make protein-protein interactions with RNA polymerase that are required for transcription activation at both class I and class II SoxS-dependent promoters. (5) Mutants F74A, D75A, M78A, D79A and Q85A appear to define a surface required for protein-protein interaction with RNA polymerase specifically at class II promoters because these positive control mutants bind and bend DNA normally but are defective in activation of class II promoters but not class I promoters. These SoxS mutants that bind and bend DNA normally but are defective in transcription activation represent the first positive control mutants with putative defects in protein-protein interactions with RNA polymerase among the SoxS/MarA/Rob subset of the AraC/XylS family of transcription regulators.
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PMID:A comprehensive alanine scanning mutagenesis of the Escherichia coli transcriptional activator SoxS: identifying amino acids important for DNA binding and transcription activation. 1221 88

We have investigated protein-protein interactions among the respiratory syncytial virus (RSV) RNA polymerase subunits using affinity chromatography. Here we demonstrate a novel interaction of P and M2-1 proteins. Phosphorylation of either M2-1 or P appears to be dispensable for this interaction. Internal deletions within P mapped the M2-1-binding domain to a region between residues 100 and 120. Alanine-scanning mutagenesis within this region of P revealed that substitution of any one of the three residues, L101, Y102, and F109, prevented both M2-1 and P binding and expression of an M2-1-dependent luciferase reporter gene. However, these same mutations did not prevent the activity of an M2-1-independent chloramphenicol acetyltransferase minigenome, suggesting that these residues of P specifically affect M2-1-P interaction. On the basis of these observations, it is possible that the interaction between RSV M2-1 and P proteins is important for viral replication.
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PMID:Interaction between human respiratory syncytial virus (RSV) M2-1 and P proteins is required for reconstitution of M2-1-dependent RSV minigenome activity. 1297 Apr 53

The mammalian Orthoreovirus (mORV) core particle is an icosahedral multienzyme complex for viral mRNA synthesis and provides a delimited system for mechanistic studies of that process. Previous genetic results have identified the mORV mu2 protein as a determinant of viral strain differences in the transcriptase and nucleoside triphosphatase activities of cores. New results in this report provided biochemical and genetic evidence that purified mu2 is itself a divalent cation-dependent nucleoside triphosphatase that can remove the 5' gamma-phosphate from RNA as well. Alanine substitutions in a putative nucleotide binding region of mu2 abrogated both functions but did not affect the purification profile of the protein or its known associations with microtubules and mORV microNS protein in vivo. In vitro microtubule binding by purified mu2 was also demonstrated and not affected by the mutations. Purified mu2 was further demonstrated to interact in vitro with the mORV RNA-dependent RNA polymerase, lambda3, and the presence of lambda3 mildly stimulated the triphosphatase activities of mu2. These findings confirm that mu2 is an enzymatic component of the mORV core and may contribute several possible functions to viral mRNA synthesis.
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PMID:Nucleoside and RNA triphosphatase activities of orthoreovirus transcriptase cofactor mu2. 1461 38

Escherichia coli Rsd protein was previously identified on the basis of its binding to the RNA polymerase sigma(70) subunit. The Rsd-sigma(70) complex has been studied using different methods. Our data show that Rsd associates with sigma(70) to form a complex with a stoichiometry of 1:1. Alanine scanning and deletion mutagenesis were used to locate regions of sigma(70) that are required for the formation of the Rsd-sigma(70) complex.
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PMID:Studies of the Escherichia coli Rsd-sigma70 complex. 1468 66

Sinorhizobium meliloti DctD is an activator of sigma(54)-RNA polymerase holoenzyme and member of the AAA+ superfamily of ATPases. DctD uses energy released from ATP hydrolysis to stimulate the isomerization of a closed promoter complex to an open complex. DctD binds to upstream activation sequences (UAS) and contacts the closed complex through DNA looping to activate transcription, but the UAS is not essential for activation if DctD is expressed at higher than normal levels. Introduction of specific substitutions within or near the conserved ESELFG motif in the C3 region of a truncated, constitutively active form of DctD produced several mutant forms of the protein that had increased dependence on the UAS for activation. Removing the DNA-binding domain from one UAS-dependent mutant and from one activation-deficient mutant significantly increased transcriptional activation, indicating that the DNA-binding domain interfered with the activities of these mutant proteins. A UAS-dependent mutant with a P315L substitution in the C6 region was identified from a genetic screen. Alanine scanning mutagenesis of conserved amino acid residues around Pro-315 produced two additional UAS-dependent mutants as well as several mutants that failed to activate transcription but retained ATPase activity. In contrast to the two mutant proteins with substitutions in the C3 region, removal of the DNA-binding domain from the mutant proteins with substitutions in the C6 region did not stimulate their activity. The residues in the C6 region that were altered are in a probable hinge region between the alpha/beta and alpha-helical subdomains of the AAA+ domain. The alpha-helical subdomain contains the sensor II helix that has been implicated in other AAA+ proteins as sensing changes in the nucleotide during the hydrolysis cycle. Substitutions in the hinge region may have abolished nucleotide sensing by interfering with subdomain interactions, altering the relative orientation of the sensor II helix or interfering with oligomerization of the protein.
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PMID:Novel substitutions in the sigma54-dependent activator DctD that increase dependence on upstream activation sequences or uncouple ATP hydrolysis from transcriptional activation. 1545 3


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