Stimulation of open complex formation by nicks and apurinic sites suggests a role for nucleation of DNA melting in Escherichia coli promoter function.
We report the effects of depurination and prenicking at various positions of the phage lambda prmup-1Delta265 promoter DNA on the rate of open complex formation. We have found that depurination and prenicking at positions around the -10 region strongly stimulated the rate of open complex formation. Since nicking and depurination are known to destabilize DNA helical structure, our observations indicate that the instability of the -10 region is important for open complex formation. We further infer that (i) the nucleation of DNA melting, which occurs during the isomerization from the closed complex into the open complex, contributes to the rate of open complex formation; (ii) the nucleation of melting occurs around the -10 region; and (iii) the propagation of DNA melting from the nucleation region is not rate-limiting. In addition, we have found that depurination at several positions inhibited open complex formation. We used dimethyl sulfate modification protection studies to show that most of the guanine bases that are among these positions are in contact with RNA polymerase in the open complex.
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Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
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D001483:Base Sequence; D004249:DNA Damage; D018983:DNA Footprinting; D004279:DNA, Viral / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011685:Purine Nucleotides; D013463:Sulfuric Acid Esters; D014158:Transcription, Genetic
D004279:DNA, Viral; D011685:Purine Nucleotides; D013463:Sulfuric Acid Esters; D012321:DNA-Directed RNA Polymerases; C007482:dimethyl sulfate