Influence of the linking number of template DNA on σ³²-dependent transcription initiation in vitro of the Escherichia coli groE gene

Plasmids carrying the groE promoter with a superhelical density of 0 to -0.091 were used for in vitro transcription with RNA polymerase holoenzyme containing σ³². The promoter activity of the groE gene in vitro on the plasmid DNA was decreased to 100-fold when the superhelical density of the template DNA decreased from -0.059 to 0. As the superhelical density of DNA inside the cells was estimated to be approximately -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 DNA. At higher concentrations of salt, the dependence of the groE promoter activity on negative supercoiling was even more apparent. Since melting of the DNA duplex is inhibited by high salt concentrations, DNA supercoiling may facilitate melting of the groE promoter by an RNA polymerase holoenzyme containing σ³².