Recognition of the -10 promoter sequence by a partial polypeptide of sigma70 in vitro

Promoter recognition by RNA polymerase depends upon its ability to bind to specific DNA sequences. The sigma (sigma) subunit provides selectivity for transcription initiation by interacting with the -10 and -35 elements of promoter DNA. Suppressor mutations in sigma factor that compensate for specific "down" substitutions in the promoter have demonstrated that sigma factor recognizes certain base pairs of the promoter. Since these suppressors were only identified for changes at the -12 and -11 positions of the -10 element (TATAAT), the role of the other base pairs of this region in specifying recognition by sigma factor remained unclear. Using a partial polypeptide of sigma70 carrying regions 2-4, this report shows that the first three positions of the -10 element (-12, -11, -10) are important for sigma factor alone to recognize and bind to duplex DNA. The sigma polypeptide also binds to an "extended -10" promoter, even without a -35 element. A mismatch bubble from -10 to +3 is bound regardless of the sequence within the bubble, or the presence or absence of a -35 element. Unexpectedly, binding to a mismatch bubble that lacks a -35 hexamer is sensitive to the identity of the -11 position, but not the -12 position.