Comparison of the open complexes formed by RNA polymerase at the Escherichia coli lac UV5 promoter.
In transcription initiation at the lac UV5 promoter, Escherichia coli RNA polymerase forms two open complexes, called Ou and O1, which can be separated by electrophoresis on native polyacrylamide gels. We have compared the properties of these two open complexes, with the objective of rationalizing the functional difference previously reported between the two forms: the complex which is dominant at high temperature (Ou) is better able to escape abortive transcriptional cycling into productive mRNA elongation. Methylation protection and binding domain probing with exonuclease III were used to investigate differences in polymerase binding strength to particular DNA domains. Also, we examined the difference in the extent and temperature dependence of promoter unwinding in the two complexes, as probed by methylation of unpaired cytosines and cleavage by phage T7 endonuclease. We find that O1 has stronger promoter interactions in the DNA domain whose upstream edge is defined by an exonuclease III stop at -24. These -24 domain interactions, which presumably aid in promoter binding and nucleation of DNA unwinding, are inferred to be strong enough to hinder escape of the polymerase from the open complex contacts that are maintained during abortive initiation. The Ou complex has weaker binding to the -24 domain, partially compensated by better upstream interactions and a better ability to accommodate extensive DNA unwinding. It thus escapes abortive initiation more readily because of weaker critical open complex contacts that must be lost when stable initiation occurs from the corresponding stressed intermediates.
D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D046911:Macromolecular Substances; D008745:Methylation; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000378:metabolism; D013463:Sulfuric Acid Esters; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D046911:Macromolecular Substances; D012329:RNA, Bacterial; D013463:Sulfuric Acid Esters; D012321:DNA-Directed RNA Polymerases; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C007482:dimethyl sulfate