Studies on the interaction of T7 RNA polymerase with a DNA template containing a site-specifically placed psoralen cross-link. I. Characterization of elongation complexes.
A 66 base-pair (bp) DNA template carrying a site-specifically placed psoralen cross-link downstream from a phage T7 promoter was constructed. This template can support transcription by T7 RNA polymerase. Transcription was blocked specifically at the psoralen cross-link. We studied the characteristics of elongation complexes, formed in this manner, by enzymatic and chemical footprinting and by a nitrocellulose filter-binding assay. The DNase I footprint of the elongation complex was quantified on a residue by residue basis. It was found that T7 RNA polymerase made the strongest contacts in the central region of the footprint whereas the leading and lagging edges of the polymerase were weakly bound to the DNA. Reducing the NaCl concentration in the transcription reaction resulted in the visualization of two T7 RNA polymerase molecules bound to the same template. A leading polymerase molecule, arrested at the psoralen cross-link, showed a much smaller DNase I footprint than a lagging polymerase molecule that was bound upstream. This upstream polymerase molecule occupied approximately one-half of the promoter region and therefore did not achieve complete promoter clearance. These experiments suggest that complete promoter clearance is required for a gross conformational change in the polymerase, consisting of a contraction in the size of its footprint to occur. DNase I footprinting also revealed that an elongation complex arrested at a psoralen cross-link undergoes several subtle changes in structure in a time-dependent manner and therefore can be considered to be in a state of dynamic flux. Methylation protection showed that some G residues in the top (non-coding) strand are protected against attack by dimethylsulfate, whereas the G residues on the bottom (coding) strand appear not to be protected from reaction with dimethylsulfate. We probed the transcribing complexes for single-stranded regions with T7 gene 3 endonuclease. From the pattern of sensitivity to T7 gene 3 endonuclease on the template strand, we conclude that the RNA-DNA hybrid in the elongation complex is about 7 bp. A nitrocellulose filter-binding assay showed that the elongation complex, consisting of a 36 (+1) nucleotide RNA, the 66 bp DNA template and the T7 RNA polymerase was stable for at least 30 minutes at high salt concentrations. Heparin caused the quantitative release of 36 (+1) RNA nucleotides within 30 seconds, but the DNA was not simultaneously released from the elongation complex under these conditions.
Department of Chemistry, University of California, Berkeley.
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Sastry
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Hearst
D001483:Base Sequence; D004279:DNA, Viral / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008745:Methylation; D008969:Molecular Sequence Data; D012965:Sodium Chloride / Q000494:pharmacology; D013604:T-Phages / Q000201:enzymology / Q000235:genetics; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D014307:Trioxsalen / Q000031:analogs & derivatives / Q000737:chemistry; D014764:Viral Proteins
D004279:DNA, Viral; D046911:Macromolecular Substances; D014764:Viral Proteins; C016507:hydroxymethyltrioxsalen; D012965:Sodium Chloride; D006493:Heparin; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D014307:Trioxsalen