Roles for Inhibitory Interactions in the Use of the–10 Promoter Element by σ70 Holoenzyme

A panel of seven -10 region DNA mutants was tested for holoenzyme binding against a panel of 13 region 2 mutants of sigma 70. No patterns were noticed that would indicate unique interactions between individual amino acids and individual -10 region bases. Instead, certain amino acid substitutions led to increased holoenzyme binding to DNA, implying that the wild type interactions are associated with an inhibitory component. These inhibitory interactions were stronger on DNA containing non-consensus sequences, like those of typical promoters. In addition, the DNA segment downstream from the -10 element was also inhibitory to binding when in duplex form but stimulated binding when in single strand form. Overall, the data suggest that -10 region duplex recognition and melting have a large component of overcoming unfavorable protein:DNA base interactions, particularly when the bases are non-consensus, and that this contributes to setting physiologically appropriate variations in transcription rate.