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)

We have charted the movements of E sigma 32 RNA polymerase at the heat-shock promoter PgroE throughout open complex formation, using hydroxyl radical footprinting. In combination with methylation protection and DNase I experiments, these data suggest the following model for open complex formation. E sigma 32 initially anchors itself in the upstream region of the promoter forming the first closed complex, RPC1; in this complex the enzyme makes backbone contacts in the -35 region of the promoter that are maintained throughout open complex formation. An isomerization follows resulting in a second closed complex, RPC2; in this complex the enzyme makes base-specific and backbone contacts in the -10 region that are almost identical to those found in the open complex. Thus, at the groE promoter, upstream contacts are established in RPC1 and downstream contacts in RPC2. A similar pattern of backbone contacts was obtained for E sigma 32 bound in the open complex at two additional heat-shock promoters, suggesting that the overall topology of holoenzyme in the open complex is similar regardless of sequence variations in the promoter.
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PMID:Development of RNA polymerase-promoter contacts during open complex formation. 165 95

Simian virus 40 (SV40) T antigen stimulates the level of transcription from several RNA polymerase II promoters, including the SV40 late promoter. The mechanism of trans activation appears to be indirect since binding of T antigen to specific DNA sequences is not required. However, specific promoter elements that respond to T antigen have not previously been defined. We identified DNA sequences from the SV40 late promoter whose ability to stimulate transcription is induced by the expression of T antigen. In particular, the Sph I + II motifs of the SV40 enhancer can confer T-antigen inducibility to the normally uninducible herpes simplex virus thymidine kinase gene promoter when multiple copies of the sequence are inserted 5' of the transcription initiation site and TATA sequence. Binding sites for the cellular transcription factor TEF-1 and octamer binding proteins are contained within the Sph I + II motifs, as well as at other positions in the SV40 promoter. To study the role of individual protein-binding sites in trans activation by T antigen, mutations were constructed in various TEF-1 and octamer protein-binding sites of the SV40 late promoter. These mutations did not significantly affect basal promoter activity. However, mutation of all three TEF-1 sites prevented detectable activation by T antigen. DNase I footprinting of the mutated promoters with purified proteins demonstrated that inducibility by T antigen correlated with binding affinity of TEF-1 for the DNA and not with binding affinity of an octamer binding protein.
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PMID:trans activation of the simian virus 40 late promoter by large T antigen requires binding sites for the cellular transcription factor TEF-1. 165 59

Some properties of DNA condensed with spermidine have been compared with the properties of DNA condensed with Co3+(NH3)6 to determine whether condensation of DNA with these trivalent cations protects DNA against the action of DNase I and increases transcription and encapsulation of DNA into liposomes. It was shown that DNA condensed with Co3+(NH3)6 was resistant to the action of the endonuclease DNase I such as DNA condensed with spermidine was. However, DNA condensed with Co3+(NH3)6 was significantly less active in transcription with the E. coli RNA polymerase than DNA-spermidine condensed forms. In addition, it was demonstrated that both compacted forms of DNA were more efficiently encapsulated into neutral liposomes; however, negatively, charged liposomes were scarcely formed in the presence of DNA condensed with Co3+(NH3)6. These experiments and the well documented properties of polyamines increasing the resistance to radiations and hydrolysis of nucleic acids, as well as their biological activities, such as replication, transcription, and translation, together with the low concentration of Co3+ in the environment, lead us to propose spermidine as a plausible prebiotic DNA condensing agent rather than Co3+ and the basic proteins proposed by other authors. Then, we consider the possible role and relevance of the polyamine-nucleic acids complexes in the evolution of life.
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PMID:Possible prebiotic significance of polyamines in the condensation, protection, encapsulation, and biological properties of DNA. 166 78

Binding of high mobility group (HMG) proteins 14 and 17 (HMG 14/17) to complete nucleosomal cores and to cores lacking one H2A.H2B dimer, the amino-terminal tails of histones, or both one H2A.H2B dimer and the amino-terminal ends of histones is accompanied by an overall stabilization of the particles as determined by thermal denaturation, circular dichroism and DNase I digestion. In spite of the structural stabilization brought about by HMG 14/17, the presence of these proteins causes little effect on the efficiency of the different nucleosomal particles as transcription templates for RNA polymerase II. The nucleosomal particles lacking one H2A.H2B dimer and containing two bound HMG 14/17 molecules are efficient in vitro transcription templates, which allow transcription of the whole length of the DNA present in the particle. These results are consistent with HMG 14/17 being present in active chromatin.
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PMID:Structural and transcriptional properties of different nucleosomal particles containing high mobility group proteins 14 and 17 (HMG 14/17). 169 25

In this study we describe DNA-RNA complexes in matrix DNA of Friend cells. The presence of such unusual structures is confirmed by the following evidence. When a preparation of matrix DNA is electrophoresed in agarose an RNA component always migrates together with DNA. There should be a close interaction between DNA and RNA in such a preparation because the presence of the RNA component causes resistance of DNA to DNase I and Exo III. An intimate, hybrid-type association of part of the RNA component with DNA is indicated also by the fact that about 20% of this RNA is sensitive to RNase H. By specific inhibition of the RNA synthesis with alpha-amanitin and actinomycin D it was shown that the bulk of associated RNA is transcribed by RNA polymerase III. Hybridization experiments showed similarity between the DNA sequences isolated from the complexes and those from the base of dehistonized DNA loops obtained by high-salt extraction of nuclei. This observation suggests that the complexes might represent attachment sites of nuclear DNA to the matrix: possibly, the attachment is mediated via the RNA component. Experiments with induction of erythroid differentiation indicated that a profound reorganization of the nucleus, accompanying terminal differentiation, leads to a striking reduction in the number of complexes and thus in the number of attachment sites. This suggests that the complexes should function as transient attachment sites.
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PMID:DNA-RNA complexes that might represent transient attachment sites of nuclear DNA to the matrix. 169 80

We have purified specific RNA polymerase II elongation intermediates initiated at the adenovirus type 2 major late promoter and paused either 15 or 35 to 36 bases downstream of the transcription initiation site. Transcription was arrested at these two sites by combining modification of the promoter sequence with limitation of appropriate nucleotide concentrations in the in vitro reaction. The resultant complexes were remarkably stable and could be purified away from free DNA and contaminating protein-DNA complexes, without loss of activity, by the use of sucrose gradient sedimentation and low-ionic-strength polyacrylamide gel electrophoresis. The complexes were characterized by both DNase I and o-phenanthroline-copper ion nuclease protection assays. The DNase I footprints revealed that the structures of the 15- and 35- to 36-nucleotide transcription complexes differed from those previously reported for an adenovirus type 2 major late preinitiation complex and a subsequent intermediate formed upon addition of ATP. Furthermore, the 35- to 36-nucleotide complex protected a significantly smaller portion of the template than the 15-nucleotide species and migrated at a slightly higher rate in polyacrylamide gels. These observations suggest that changes in structural organization may continue to occur in transcription complexes which are already committed to elongation.
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PMID:RNA polymerase II elongation complexes paused after the synthesis of 15- or 35-base transcripts have different structures. 170 7

The E. coli thr operon leader region contains a cluster of transcription pause sites upstream of the attenuator. In this report, we determine the exact sites of pausing and analyze the structure of the ternary complex by footprint techniques. Under synchronized transcription initiation conditions in vitro, three closely-spaced transcription pause sites were identified. These pause sites appeared downstream of the first region of dyad symmetry, which encodes an RNA hairpin in the transcript, and occurred at positions G112, G114 and A116 of the thr leader RNA. The results showed that the half-life of the thr paused complexes at G112 and G114 could be enhanced by limiting the concentration of the nucleoside triphosphate GTP in the transcription reactions. In addition, the half-life of the paused complexes was shown to increase in the presence of NusA protein. The thr leader complex that paused immediately before residues G112 and G114 of the nascent transcript was isolated and its structure was analyzed with enzymatic and chemical cleavage reagents. The footprinting studies using DNase I showed that there were approximately 35 nucleotides on both strands of the DNA that were protected by RNA polymerase from DNase I cleavage. The DNA segment protected by RNA polymerase is approximately 19 nucleotides upstream and 14 nucleotides downstream of the pause sites. The results from hydroxyl radical footprints also showed a similar pattern of protection at the transcription pause sites. However, no significant differences in the footprinting patterns were observed in the presence or absence of NusA protein.
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PMID:Isolation and footprint analysis of the Escherichia coli thr leader paused transcription complex. 170 79

Transcription of the Escherichia coli crp gene encoding cAMP receptor protein (CRP) is negatively regulated by CRP-cAMP complex that binds to a specific site located downstream from the transcription start site. The binding of CRP-cAMP to this site activates transcription from a second divergent overlapping promoter. The mechanism of this negative autoregulation of the crp gene has been investigated by in vitro transcription, gel shift, DNase I footprinting, and exonuclease III protection assays. We demonstrated that the crp and divergent promoters are reciprocally and coordinately regulated by CRP-cAMP. The abortive initiation assay revealed that the divergent RNA itself is not required for the inhibition of crp transcription. Detailed binding studies revealed that CRP-cAMP stimulates the binding of RNA polymerase to the divergent promoter and thus blocks the occupation of the crp promoter by RNA polymerase.
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PMID:Molecular mechanism of negative autoregulation of Escherichia coli crp gene. 171 82

In vitro, Pseudomonas aeruginosa TrpI protein activates transcription initiation at the trpBA promoter (trpPB) and represses initiation at its own promoter (trpPI), which diverges from, and overlaps, trpPB. Indoleglycerol phosphate (InGP) reduces the TrpI concentration required for binding to its strong binding site (site I), as measured by repression of trpPI; it also facilitates activation of trpPB, presumably because it enables TrpI to bind to a weaker binding site (site II) and thereby interact with RNA polymerase. The role of site II and InGP in regulation of the two promoters was investigated by constructing site II mutants. A 2 bp substitution affected the ability of TrpI to activate trpPB, but did not significantly affect TrpI binding to site II. A more extensive (8 bp) substitution inhibited TrpI-mediated activation of trpPB and TrpI-mediated protection of site II in a DNase I footprinting assay. However, the mutation did not alter the pattern of TrpI binding observed in gel retardation experiments. In particular, a more slowly-migrating complex (Complex 2) whose appearance was correlated with TrpI binding to site II was formed equally well on a wild-type or substituted DNA fragment. Based on the mutant phenotypes, we propose that a particular sequence of protein--protein and protein--DNA interactions is required for activation of trpPB by TrpI and InGP.
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PMID:Mutations in TrpI binding site II that differentially affect activation of the trpBA promoter of Pseudomonas aeruginosa. 175 20

The Bacillus subtilis dciA operon, which encodes a dipeptide transport system, was induced rapidly by several conditions that caused the cells to enter stationary phase and initiate sporulation. The in vivo start point of transcription was mapped precisely and shown to correspond to a site of transcription initiation in vitro by the major vegetative form of RNA polymerase. Post-exponential expression was prevented by a mutation in the spo0A gene (whose product is a known regulator of early sporulation genes) but was restored in a spo0A abrB double mutant. This implicated AbrB, another known regulator, as a repressor of dciA. In fact, purified AbrB protein bound to a portion of the dciA promoter region, protecting it against DNase I digestion. Expression of dciA in growing cells was also repressed independently by glucose and by a mixture of amino acids; neither of these effects was mediated by AbrB.
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PMID:Transcriptional regulation of a Bacillus subtilis dipeptide transport operon. 176 71


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