Thermal energy requirement for strand separation during transcription initiation: the effect of supercoiling and extended protein DNA contacts

We have studied the role of extended protein DNA contacts and DNA topology on the ability of Escherichia coli RNA polymerase to form open complexes at several related promoters. The -35 region of several Escherichia coli promoters do not have homology with the consensus sequence, but still drive activator independent transcription initiation. This is due to the presence of a TG motif upstream from the -10 hexamer creating an 'extended -10' promoter. We have previously shown that two 'extended -10' promoters, galP1 and pBla, can form open complexes at lower temperatures than the galP1 derivative, galPcon6, which has a consensus -35 hexamer. Here we report further investigations into the mechanism of open complex formation by RNA polymerase, in particular the thermal energy requirement. A single base pair change in galPcon6 creating an 'extended -10' sequence, results in a 20 degrees C reduction in the temperature requirement for open complex formation. The DNA topology has also been shown to effect the thermal energy requirement for strand separation. Promoters carried on supercoiled plasmids form open complexes at lower temperatures than when present on linear DNA templates. We have also shown that in vivo, RNA polymerase can form open complexes at lower temperatures than those observed for linear templates in vitro, but requires slightly higher temperatures than supercoiled templates in vitro, however the promoter hierachy remains the same.