CONTRIBUTIONS OF PROMOTER CONTEXT AND STRUCTURE TO REGULATED EXPRESSION OF THE F-PLASMID TRAY PROMOTER IN ESCHERICHIA-COLI K-12

Expression of the F plasmid traY promoter in vivo requires both host ( E. coli) and plasmid encoded proteins. As judged by transcript size an d primer extension analyses, the F plasmid traY promoter was utilized in vitro by purified E. Coli sigma70 RNA polymerase in the absence of other proteins. However, in vitro transcription required supercoiled t emplates. Endonuclease protection experiments showed that RNA polymera se is unable to form a stable complex at the traY promoter in linear o r relaxed circular templates. In vitro transcription with linear templ ates could be elicited by altering the traY-10 and -35 hexamers to the consensus sequences. Alterations that reduced the effect of template supercoiling on apparent promoter strength in vitro also reduced the e ffect of the F plasmid TraJ protein on traY expression in vivo. Appare nt traY promoter strength in vitro, estimated in template competition experiments, was unaltered by deletion of tra DNA normally upstream of the promoter, a change in promoter context that elicited high levels of promoter activity in TraJ- cells. These data suggest a model for re gulated traY promoter activity in which a nucleoprotein complex involv ing tra DNA immediately upstream locally relaxes traY promoter DNA. Tr aJ and perhaps other activators could disrupt the complex, allowing pr omoter DNA to equilibrate at the prevailing negative superhelical dens ity and thereby eliciting transcription initiation.