Effect of DNA bases and backbone on σ70 holoenzyme binding and isomerization using fork junction probes

Abasic substitutions in the non-template strand and promoter sequence changes were made to assess the roles of various promoter features in sigma70 holoenzyme interactions with fork junction probes. Removal of -10 element non-template single strand bases, leaving the phosphodiester backbone intact, did not interfere with binding. In contrast these abasic probes were deficient in promoting holoenzyme isomerization to the heparin resistant conformation. Thus, it appears that the melted -10 region interaction has two components, an initial enzyme binding primarily to the phosphodiester backbone and a base dependent isomerization of the bound enzyme. In contrast various upstream elements cooperate primarily to stimulate binding. Features and positions most important for these effects are identified.