THERMAL-ENERGY REQUIREMENT FOR STRAND SEPARATION DURING TRANSCRIPTIONINITIATION - THE EFFECT OF SUPERCOILING AND EXTENDED PROTEIN-DNA CONTACTS

We have studied the role of extended protein DNA contacts and DNA topo logy on the ability of Escherichia coil RNA polymerase to form open co mplexes at several related promoters. The -35 region of several Escher ichia coil 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 cre ating an 'extended -10' promoter. We have previously shown that two 'e xtended -10' promoters, galP1 and pBla, can form open complexes at low er temperatures than the galP1 derivative, galP(con)6, which has a con sensus -35 hexamer. Here we report further investigations into the mec hanism of open complex formation by RNA polymerase, in particular the thermal energy requirement. A single base pair change in galP(con)6 cr eating an 'extended -10' sequence, results in a 20 degrees C reduction in the temperature requirement for open complex formation. The DNA to pology has also been shown to effect the thermal energy requirement fo r strand separation. Promoters carried on supercoiled plasmids form op en complexes at lower temperatures than when present on linear DNA tem plates. We have also shown that in vivo, RNA polymerase can form open complexes at lower temperatures than those observed for linear templat es in vitro, but requires slightly higher temperatures than supercoile d templates in vitro, however the promoter hierachy remains the same.