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)

We have defined protein-DNA interactions associated with the transcription complex of a Xenopus somatic 5 S RNA gene under efficient in vitro transcription conditions. Transcription factor IIIA, the internal control region of the 5 S RNA gene and specific DNA sequences 5' and 3' of the internal control region are all involved in the formation of a multiprotein complex. Specific protein-DNA interactions outside of the 5 S RNA gene itself, revealed by DNase I footprinting, have no apparent role in the transcription process. Sequences within the 5 S RNA gene, 5' of the internal control region, are not essential for transcription by RNA polymerase III in vitro, but do contribute to its efficiency.
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PMID:The transcription complex of the Xenopus somatic 5 S RNA gene. A functional analysis of protein-DNA interactions outside of the internal control region. 230 78

We have analysed the transcription of a functional human 7SL gene by RNA polymerase III (RNAPIII) in S100 extracts in vitro. Accurate and efficient synthesis of 7S L RNA depends on the presence of (i) an upstream sequence and (ii) an internal promoter element located within the first 22 bp of the gene. These findings were substantiated by DNase I footprinting. Mutations of the internal promoter identified the doublet CG [nucleotide (nt) +15/+16] outside the A-box homologue (nt +5 to +14) as being essential for both proper promoter function in the in vitro transcription assay and competition in the template-exclusion assay. Fractionation of S100 extracts identified two fractions required in addition to RNAPIII for faithful transcription of the gene. Each of these two fractions gave rise to one of two footprints observed in DNase I protection experiments, indicating that at least two DNA-binding factors are involved.
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PMID:Sequence and factor requirements for faithful in vitro transcription of human 7SL DNA. 232 74

Homopolymeric dAn.dTn sequences, where n is 4 or greater, have special properties leading to increased duplex stability and DNA bending. The lacUV5 promoter was used to examine the functional consequences of changing the -10 TATAAT consensus sequence to the sequence TAAAAT. The transversion mutation at the underlined site was accomplished with site-directed mutagenesis using translation termination as the selection procedure. For free DNA, structural differences at the 5' and 3' junction regions of the dA4.dT4 tract can be readily detected by DNase I digestion. However, site binding by Escherichia coli RNA polymerase appeared unaltered by the TAAAAT sequence since identical DNase I footprints were obtained for the lacUV5 and mutant promoters. Binding competition studies under different ionic strengths revealed a significant reduction in mutant promoter open complex formation relative to the lacUV5 promoter. Mutant promoter open complexes also dissociated faster and to a greater extent than the corresponding lacUV5 promoter open complexes when challenged with heparin or a combination of heparin and increased KCl concentration. Consequently, mutant promoter open complexes appear less stable than lacUV5 promoter open complexes.
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PMID:Site-directed mutagenesis of the -10 region of the lacUV5 promoter. Introduction of dA4.dT4 tract suppresses open complex formation. 240 69

We have used enzymatic and chemical probes to follow the movement of Escherichia coli RNA polymerase along lacUV5 promoter DNA during transcription initiation. The RNA polymerase does not escape from the promoter but remains tightly bound during the synthesis of the initial bases of the transcript. This initial phase of RNA synthesis involves the reiterative synthesis and release of RNA chains up to ten bases long via the RNA polymerase cycling reaction and the enzyme remains sensitive to rifampicin inhibition. When longer chains are made, promoter-specific binding is disrupted and the enzyme forms a rifampicin-resistant elongation complex with downstream DNA sequences. This elongation complex covers less than half as much DNA and lacks the DNase I-hypersensitive sites and the base-specific contacts that characterize promoter-bound RNA polymerase. These results lead us to suggest that lacUV5 mRNA synthesis is primed by a promoter-bound enzyme complex that synthesizes the initial nine or ten bases in the mRNA chain. Subsequently, when a chain of ten bases, or slightly longer, is made, contacts with promoter DNA are irreversibly disrupted, sigma subunit is lost, and a "true" elongation complex is formed.
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PMID:Interaction of RNA polymerase with lacUV5 promoter DNA during mRNA initiation and elongation. Footprinting, methylation, and rifampicin-sensitivity changes accompanying transcription initiation. 240 92

We have prepared three types of RNA polymerase II transcription complexes: a preinitiation complex (complex 0), a complex which has synthesized two phosphodiester bonds (complex 2), and a complex which has synthesized 10-13 bonds (complex 10). We have studied the differential response of these complexes to a variety of disruptions: detergent (Sarkosyl), high levels of KCl, extended incubation at 25 degrees C, proteolysis, and digestion with DNase I. Complex 0 is extremely stable at 25 degrees C in the absence of ATP, but it is sensitive to the other treatments including 25 degrees C incubation in the presence of ATP. Once the complex has made two phosphodiester bonds, the properties almost reverse from those of complex 0; complex 2 remains unstable at 25 degrees C in the presence of ATP but is resistant to high levels of Sarkosyl and KCl, to extensive DNase I digestion, and to brief proteolysis. Addition of 10 or more bases to the growing RNA chain results in a complex completely resistant to all of the treatments used. When DNase I-trimmed complex 0 is allowed to initiate RNA synthesis, chains of about 33 bases are obtained. In contrast, DNase-trimmed complex 2 gives only about 23 base transcripts; DNase-treated complex 10 will elongate its nascent chains by about 21 bases as well (to give, on average, 34 base transcripts).
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PMID:Transcription initiation by RNA polymerase II in vitro. Properties of preinitiation, initiation, and elongation complexes. 243 61

Xenopus transcription factor IIIA (TFIIIA) is expressed in Escherichia coli by utilizing one plasmid with a T7 RNA polymerase gene and another plasmid with TFIIIA cDNA cloned downstream of a T7 promoter. Wild-type TFIIIA and a TFIIIA deletion mutant, isolated from E. coli cell extracts, are identified by antiserum against native TFIIIA purified from Xenopus immature oocytes. DNase I protection experiments indicate that wild-type TFIIIA, synthesized from a full-length TFIIIA cDNA, binds specifically to the coding and noncoding strands of the 5 S RNA gene. The TFIIIA deletion mutant, expressed from TFIIIA cDNA lacking the coding sequence for the N-terminal 29 amino acids, fails to bind specifically to the 5 S RNA gene as judged by its inability to protect to any degree the coding or noncoding strands of the gene from DNase I digestion. Both wild-type TFIIIA and the N-terminal deletion mutant promote DNA renaturation.
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PMID:Deletion of the N-terminal region of Xenopus transcription factor IIIA inhibits specific binding to the 5 S RNA gene. 244 98

The properties of the two monoclonal antibodies which were found to inhibit cyclic AMP receptor protein (CRP)-stimulated abortive initiation without affecting cAMP binding (Li, X.-M., and Krakow, J. S. (1986) J. Biol. Chem. 260, 4378-4383) have been characterized. Binding of monoclonal antibody (mAb) 66C3 to CRP is stimulated by cAMP while CRP binding by mAb 63B2 is not affected by cAMP. Binding of cAMP-CRP-mAb 63B2 to the lac P+ DNA is completely inhibited. Whereas cAMP-CRP forms a stable complex only at the CRP site 1 of the lac P+ promoter fragment, cAMP-CRP-mAb 66C3 binds to both site 1 and site 2. DNase I footprinting using a HpaII fragment carrying only the lac site 2 does not show any protection by cAMP-CRP-mAb 66C3. With the lac L8UV5 promoter, binding is not seen at either the L8 site 1 or the unaltered site 2. In the presence of 25% glycerol, cAMP-CRP-mAb 66C3 binds to both L8 site 1 and site 2. RNA polymerase is unable to bind to the cAMP-CRP-mAb 66C3-lac P+ complex. In the presence of RNA polymerase, cAMP-CRP forms a stable complex at the L8 site 1, the subsequent addition of mAb 66C3 results in the release of CRP. The CRP present in the lac P+ open promoter complex is partially resistant to subsequent incubation with mAb 66C3. The results provide further evidence regarding possible contacts between CRP and RNA polymerase involved in establishing the open promoter complex.
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PMID:Monoclonal antibodies that inhibit activation of transcription by the Escherichia coli cyclic AMP receptor protein. 244 41

We have studied the properties and structures of a series of Escherichia coli RNA polymerase ternary complexes formed during the initial steps of RNA chain initiation and elongation. Five different templates were used that contained the bacteriophage T7 A1 promoter or the E. coli Tac or the lac UV5 promoter, as well as variant templates with alterations in the initial transcribed regions. The majority of ternary complexes bearing short transcripts (from two to nine nucleotides) are highly unstable and cannot be easily studied. This includes transcripts from the phage T7 A1 promoter, for which the stability of complexes bearing transcripts as short as four nucleotides has previously been postulated. However, with one Tac promoter template, RNA polymerase forms ternary complexes with transcripts as short as five nucleotides that are stable enough for biochemical study. We describe several approaches to identifying and isolating such stable complexes and show that stringent criteria are needed in carrying out such experiments if the results are to be meaningful. Deoxyribonuclease I (DNase I) footprinting has been used to probe the general structure of the stable ternary complexes formed as the polymerase begins transcription and moves away from the start site. The enzyme undergoes a sequence of structural changes during initiation and transition to an elongating complex. Complexes with five to eight nucleotide transcripts, designated initial transcribing complexes (ITC), have identical footprints; they all retain the sigma factor and have a slightly extended DNase I footprint (-57 to +24) as compared to the open promoter complex (-57 to +20). ITC complexes all show a region of marked DNase I hypersensitivity in the -25 region that may reflect bending or distortion of the DNA template. Complexes with 10 or 11 nucleotide transcripts, designated initial elongating complexes (IEC), have lost the sigma factor and have a slightly reduced and shifted DNase I footprint (-32 to +30). However, these IEC have not yet achieved the much smaller footprint (approximately 30 bp) reported as characteristic of elongating ternary complexes bearing longer RNA chains. During the initial phase of transcription, the RNA polymerase does not move monotonically along the DNA template as RNA chains are extended, but instead, the upstream and downstream contacts remain more or less fixed as the nascent transcript is elongated up to about eight nucleotides in length. Only after incorporation of 10 nucleotides is there significant movement of the enzyme away from the promoter region and a commitment to elongation.
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PMID:RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes. 248 70

The expression of the Bacillus subtilis phage phi 29 DNA is controlled by the viral gene 4 product, which is required for the initiation of transcription at the unique late promoter A3. Protein p4 binds specifically to a phi 29 DNA fragment containing the A3 promoter. DNase I footprinting analysis has shown that the DNA binding region for protein p4 is located between nucleotides -50 and -100 relative to the transcription start site. Methylation interference assays suggest that two eight base-pair long inverted repeats located within this binding region are the protein p4 recognition sequence. These results, together with the fact that the protein p4-dependent in vitro transcription requires the B. subtilis sigma 43-RNA polymerase, indicate that protein p4 is a transcriptional activator. The protein p4 DNA recognition region is statically bent as suggested by gel retardation and chemical cleavage assays. A model of protein p4 binding to its DNA target site is proposed.
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PMID:Characterization of a new prokaryotic transcriptional activator and its DNA recognition site. 250 24

We have utilized Bacillus subtilis phage SPO-1 DNA as a model of irradiated DNA. In this phage, all thymine (Thy) residues are replaced by 5-(hydroxymethyl)uracil (5HmUra), which is a known irradiation-induced derivative of DNA Thy. SPO-1 phage is naturally devoid of other such irradiation-induced DNA lesions. DNase I activated SPO-1 phage DNA served as well as, or even better than, the control DNAs (Bacillus subtilis DNA and calf thymus DNA) as a template-primer for Escherichia coli, Micrococcus luteus, and human HL-60 cell DNA polymerases. Furthermore, the template activity of SPO-1 phage DNA was also superior when transcription with E. coli RNA polymerase was investigated. The results reported here indicated that the replacement of Thy by 5HmUra is not deleterious to template and primer functions during DNA or RNA synthesis.
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PMID:Template-primer activity of 5-(hydroxymethyl)uracil-containing DNA for prokaryotic and eukaryotic DNA and RNA polymerases. 251 77


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