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)

Yeast RNA polymerase II initiation factor b, homolog of human TFIIH, is a protein kinase capable of phosphorylating the C-terminal repeat domain of the polymerase; it possesses a DNA-dependent ATPase activity as well. The 85 kd and 50 kd subunits of factor b are now identified as RAD3 and SSL1 proteins, respectively; both are known to be involved in DNA repair. Factor b interacts specifically with another DNA repair protein, SSL2. The ATPase activity of factor b may be due entirely to that associated with a helicase function of RAD3. Factor b transcriptional activity was unaffected, however, by amino acid substitution at a conserved residue in the RAD3 nucleotide-binding domain, suggesting that the ATPase/helicase function is not required for transcription. These results identify factor b as a core repairosome, which may be responsible for the preferential repair of actively transcribed genes in eukaryotes.
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PMID:Dual roles of a multiprotein complex from S. cerevisiae in transcription and DNA repair. 826 16

The NTRC protein of enteric bacteria is an enhancer-binding protein that activates transcription by the sigma 54-holoenzyme form of RNA polymerase under nitrogen-limiting conditions. In vitro NTRC must be phosphorylated to catalyze ATP hydrolysis and activate transcription. The site of phosphorylation of NTRC from Salmonella typhimurium is Aspartate 54, which lies in the amino-terminal regulatory domain of the protein. We used site-directed mutagenesis to make "conservative" substitutions at residue 54 to alanine, asparagine, and glutamate, and examined the properties of the mutant NTRC proteins in vitro and in vivo. In vitro none of them was detectably phosphorylated, as expected if D54 is, in fact, the sole site of phosphorylation. D54A and D54N did not activate transcription of glnA but, interestingly, D54E activated constitutively. Activation by D54E was partial compared to that by phosphorylated wild-type NTRC. Combining D54A or D54N with S160F, a change in the central domain of NTRC that partially bypasses the requirement for phosphorylation, yielded doubly mutant proteins that were as active as a form carrying S160F alone, indicating that the changes in D54 did not adversely affect the function of the remainder of NTRC. Combining D54E with S160F increased the levels of constitutive ATPase activity and transcriptional activation above those of mutant NTRC proteins carrying either single change alone. We conclude that phosphorylation of aspartate 54 is required to activate NTRC and postulate that the D54E mutation mimics phosphorylation, thereby allowing NTRC to hydrolyze ATP and activate transcription. Phenotypes of mutant strains encoding NTRC proteins with substitutions at D54 indicated that phosphorylation of NTRC at position 54 was necessary for normal growth in the absence of glutamine and that such phosphorylation occurred to some extent even in the absence of NTRB.
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PMID:Glutamate at the site of phosphorylation of nitrogen-regulatory protein NTRC mimics aspartyl-phosphate and activates the protein. 833 71

Vaccinia virus early transcription factor (VETF) activates the transcription of early gene templates by the viral RNA polymerase. VETF is a heterodimeric protein that binds to transcription promoters and has an associated DNA-dependent ATPase activity. The small subunit of VETF has sequences resembling two motifs commonly found in ATPases: an A-type ATP binding motif and a DEAH box. To investigate the functional role of the ATPase activity, we have analyzed the effect of mutations in each of the putative ATPase motifs. Recombinant VETF was expressed in HeLa cells using a vaccinia virus/T7 RNA polymerase system. Simultaneous expression of both subunits of VETF was required to obtain soluble protein with promoter binding, DNA-dependent ATPase, and transcription activation functions. The mutants with altered ATPase motifs retained promoter binding activity but had no detectable ATPase activity and no ability to activate transcription. The DEAH box mutant was shown to dominantly repress transcription activation by wildtype VETF. These results indicate that the DNA-dependent ATPase activity of VETF is essential for its transcription activation function.
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PMID:The DNA-dependent ATPase activity of vaccinia virus early gene transcription factor is essential for its transcription activation function. 837 62

RNA polymerase II initiation factor delta was previously purified from rat liver and found to possess a closely associated DNA-dependent ATPase activity and a protein kinase activity capable of phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Serizawa, H., Conaway, R.C., and Conaway, J.W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7476-7480). In addition, delta's human homolog, BTF2(TFIIH), was recently shown to have an associated DNA helicase activity (Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J.H.J., Chambon, P., and Egly, J.-M. (1993) Science 259, 58-63). Here we demonstrate that initiation factor delta also possesses DNA helicase activity. In addition, we compare the properties of delta's associated CTD kinase, ATPase, and DNA helicase activities. Whereas the enzymatic properties of ATPase and DNA helicase are similar and consistent with the possibility that they could function in ATP-dependent activation of the preinitiation complex, ATPase and CTD kinase exhibit significant differences in their nucleotide specificities, responses to DNA effectors, and sensitivities to inhibitors.
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PMID:Multifunctional RNA polymerase II initiation factor delta from rat liver. Relationship between carboxyl-terminal domain kinase, ATPase, and DNA helicase activities. 839 38

Transcription of vaccinia virus early genes in vitro requires the virally encoded RNA polymerase and early transcription factor, VETF. VETF is a promoter-binding protein with DNA-dependent ATPase activity. We have investigated the functional role of VETF in transcription activation by analyzing the interaction between the RNA polymerase and promoter DNA. Using a gel shift assay, a novel protein-DNA complex was detected that required both RNA polymerase and VETF. The complex was suggested to be a transcription initiation complex by its ability to incorporate 32P-labeled nucleotides in combinations compatible with synthesis of a short RNA chain. Competition binding studies indicated that the RNA polymerase associated specifically with a viral early promoter. These experiments demonstrate that VETF activates transcription by directly recruiting the RNA polymerase to the promoter. Sedimentation analysis showed that VETF and RNA polymerase did not form a stable complex unless promoter DNA was present, indicating that protein-protein contacts are not the sole basis for initiation complex assembly. DNase I cleavage and methylation interference analyses revealed a hyperreactive site in the center of the promoter. Radiolabeling of RNA in the RNA polymerase-promoter complex did not occur when AMP-PNP (adenyl-5'-yl imidodiphosphate) was substituted for ATP, suggesting that ATP hydrolysis is required for the initiation of transcription. A model is proposed to account for these findings.
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PMID:Recruitment of vaccinia virus RNA polymerase to an early gene promoter by the viral early transcription factor. 842 51

A key event of the sporulation process in Bacillus subtilis is the asymmetric cell division that divides the developing cell into two unequal compartments. To examine the function of vegetative cell division genes in this developmental division, we isolated and characterized the B. subtilis counterpart to the Escherichia coli minicell operon minB, which governs correct placement of the division septum. Starting from the closely linked spoIVF locus, we used walking methods to isolate the region of the B. subtilis chromosome proximate to the divIVB minicell locus. DNA sequence analysis found two open reading frames whose predicted products had significant identity to the E. coli MinC cell division inhibitor and the MinD ATPase activator of MinC, and disruption of minCD function generated a minicell phenotype in B. subtilis. Notably, no homologue to the E. coli MinE topological specificity element was found in the B. subtilis minCD region. The B. subtilis min genes were part of an operon transcribed from a major promoter more than 2.5 kb upstream from minC. An internal promoter immediately upstream from minC was dependent on RNA polymerase containing sigma-H and was active at the onset of sporulation. However, neither minC nor minD function was absolutely required for sporulation and, by implication, for asymmetric septum formation.
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PMID:The minCD locus of Bacillus subtilis lacks the minE determinant that provides topological specificity to cell division. 845 76

AMP nucleosidase (EC 3.2.2.4) from Escherichia coli and AMP deaminase (EC 3.5.4.6) from bakers' yeast are proposed to regulate cellular AMP levels under allosteric control of the activator ATP and the inhibitor, PO4. Both enzymes contain catalytic sites which bind AMP and regulatory sites which bind ATP. The deduced amino acid sequences of the proteins revealed only one region of homology in which six of eight amino acids are identical. A similar sequence is found in glyceraldehyde-3-phosphate dehydrogenase, phoE, ras proteins, RNA polymerase, K(+)-ATPase, nucleolin, and other proteins expected to have nucleotide or phosphate binding properties. In the crystal structure of glyceraldehyde-3-phosphate dehydrogenase, this sequence is part of the NAD(+)-binding site. The function of these amino acids was explored with a deletion mutant of AMP nucleosidase. The protein was over-produced in a pTZ construct using the AMP nucleosidase promoter which resulted in approximately 30% of the total protein as the desired enzyme. The mutation was characterized by DNA sequence analysis and by direct analysis of the peptides using high performance liquid chromatography-mass spectrometry. Deletion of amino acids 128-135, corresponding to DGSELTLD, produced an enzyme with a 20-fold decrease in Vmax but with smaller changes in substrate saturation kinetics, activation by MgATP, inhibition by inorganic phosphate, and inhibition by the tight-binding inhibitor, formycin 5-phosphate. The deletion mutant of AMP nucleosidase exhibits hysteresis in establishing a steady-state rate of product formation which is most pronounced in the absence of MgATP. These results establish that the sequence DGSELTLD in E. coli AMP nucleosidase is not required for binding of AMP, MgATP, or inorganic phosphate. However, the mutant enzyme has a structural defect related to the polymerization state which delays the onset of catalysis and decreases the catalytic efficiency.
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PMID:Mutagenic analysis of AMP nucleosidase from Escherichia coli. Deletion of a region similar to AMP deaminase and peptide characterization by mass spectrometry. 847 16

Transcription of linearized DNA templates by SP6 RNA polymerase requires a higher concentration of ATP than of the other three nucleotides. This requirement is not shared by T7 RNA polymerase. The ATP requirement is partially relieved when the SP6 template is supercoiled but not when it is relaxed circular DNA. The effect of supercoiling is eliminated by replacement of the A.T rich sequence downstream from the SP6 promoter with a G.C rich sequence. Examination of the reaction products indicates that the ATP dependence of transcription from a linear template is not due to an ATPase activity or to the premature termination of transcription at low ATP concentration. These data suggest that the initiation of transcription by SP6 RNA polymerase requires partial denaturation of the template in the promoter-proximal region, and that this requirement can be satisfied by negative supercoiling or by increasing the ATP concentration. ATP also reduces, but does not eliminate, the abortive transcription that leads to the production of short, prematurely terminated transcripts by SP6 polymerase from supercoiled templates.
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PMID:Transcription by SP6 RNA polymerase exhibits an ATP dependence that is influenced by promoter topology. 849 6

Inactivation of Bacillus subtilis orf1177 in an otherwise Rec+ strain reduced genetic exchange and DNA repair. When the mutation was transferred into a set of recombination-deficient and repair-deficient strains, the DNA repair and recombination ability of the double or triple mutant strains was drastically reduced. B. subtilis Orf1177 protein shares substantial homology with the Escherichia coli Mdf, RecG and UvrB proteins. In vivo analysis of UV-induced mutations suggests that Orf1177 is necessary for strand-specific DNA repair, as is the case for the E. coli MFD protein. Therefore, orf1177 and Orf1177 were termed mfd gene and Mfd protein, respectively. The purified Mfd protein has a native molecular mass of 140 kDa (expected molecular mass 133 kDa). The Mfd protein is a sequence-independent DNA binding protein with weak ATPase activity. The Mfd protein was able to displace in vitro B. subtilis or E. coli RNA polymerase stalled at a lesion. Therefore, Mfd protein appears to target the transcribed strand for repair by recognizing a stalled RNA polymerase and dissociating it from the DNA. In addition, the strong recombination-deficient phenotype of mfd- rec- strains suggest that Mfd protein is involved in homologous DNA recombination.
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PMID:The Mfd protein of Bacillus subtilis 168 is involved in both transcription-coupled DNA repair and DNA recombination. 859 98

A truncated derivative of the XylR protein, which is able to constitutively activate the sigma 54-dependent Pu promoter of the TOL (toluene biodegradation) plasmid of Pseudomonas putida, has been purified to homogeneity and its various activities have been separately examined, in vitro. The truncated regulator XylR delta A was deleted of the signal reception N-terminal module present in wild-type XylR, but retained its central activation domain and the DNA binding segment, located at its C terminus. XylR delta A bound to the region -120 to -190 bp upstream of the transcription initiation site of the Pu promoter, where previous analyses have located the XylR target site. XylR delta A showed an intrinsic ATPase activity that was strongly stimulated by DNA containing the native upstream activation sequences of Pu. Both ATPase activity and ATP binding were abolished in mutant G268N in which the Walker A domain of the central module was altered. Mutant R453H lacked ATPase activity but retained the nucleotide-binding ability of the parental protein. XylR delta A was able to activate transcription in vitro with sigma 54-RNA polymerase alone, although its activity was enhanced up to 20-fold in the presence of the integration host factor protein. The requirements for activation of the Pu promoter in vitro are consistent with the view that DNA-facilitated oligomerization of the regulator for an enhanced ATPase activity is the critical event that precedes transcription initiation at sigma 54-dependent promoters. Furthermore, additional co-regulation elements seem to adjust promoter activity in vivo to the physiological status of the cells.
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PMID:In vitro activities of an N-terminal truncated form of XylR, a sigma 54-dependent transcriptional activator of Pseudomonas putida. 863 93


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