DNA deformation in nucleoprotein complexes between RNA polymerase, cAMP receptor protein and the lac UV5 promoter probed by singlet oxygen.
Singlet oxygen (1O2), generated by exciting an eosin-Tris complex with a high intensity beam of radiation at 532 nm, was used to chemically modify bases in fragments of DNA containing the lac UV5 promoter in the presence of the DNA binding proteins, RNA polymerase and CRP (cAMP receptor protein). Subsequent treatment with piperidine selectively cleaved the DNA at specific modified bases in the sequence. Using this technique we show first that the reactivity of DNA bound by CRP differs in the presence and absence of RNA polymerase. Hence the local conformation of CRP-bound DNA must change during the transition to the open complex. However, no reactivity is observed at the sites of the 40 degrees kinks described in the cocrystal structure (Steitz, 1990). Secondly we show that there is unique CRP-dependent reactivity at a specific site (position -46 on the upper strand) in the open complex. Finally, in the open complex, 1O2 also reacts with sites 90 bp upstream from the transcription start point. This reactivity is qualitatively CRP-independent. We infer that 1O2 reacts at sites where the promoter DNA is significantly distorted, and suggest that the pattern observed reflects the functional orientation of an active transcriptional complex in which the DNA is bent to form an extended loop.
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Institut Pasteur, Unité de Physicochimie des Macromolécules Biologiques (URA 1149 du CNRS), France.
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D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D016172:DNA Fingerprinting; D015342:DNA Probes; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D004801:Eosine Yellowish-(YS) / Q000737:chemistry; D007763:Lac Operon; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry*; D011401:Promoter Regions, Genetic
D002373:Cyclic AMP Receptor Protein; D015342:DNA Probes; D009698:Nucleoproteins; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D010100:Oxygen; D004801:Eosine Yellowish-(YS)