INFLUENCE OF THE LINKING NUMBER OF TEMPLATE DNA ON SIGMA(32)-DEPENDENT TRANSCRIPTION INITIATION IN-VITRO OF THE ESCHERICHIA-COLI GROE GENE

Plasmids carrying the groE promoter with a superhelical density of 0 t o -0.091 were used for in vitro transcription with RNA polymerase holo enzyme containing sigma(32). The promoter activity of the groE gene in vitro on the plasmid DNA was decreased to 100-fold when the superheli cal density of the template DNA decreased from -0.059 to 0. As the sup erhelical density of DNA inside the cells was estimated to be approxim ately -0.025, it seems likely that the increase in the linking number of template DNA caused by stress which induces heat shock response may not stimulate the groE promoter activity in vivo on the chromosomal D NA. At higher concentrations of salt, the dependence of the groE promo ter activity on negative supercoiling was even more apparent. Since me lting of the DNA duplex is inhibited by high salt concentrations, DNA supercoiling may facilitate melting of the groE promoter by an RNA pol ymerase holoenzyme containing sigma(32).