Characterization of pre-transcription complexes made at a bacteriophage T4 middle promoter: Involvement of the T4 MotA activator and the T4 AsiA protein, a sigma(70) binding protein, in the formation of the open complex

Bacteriophage T4 middle promoters have excellent matches to the -10 consensus sequence for the sigma(70) subunit of Escherichia coli RNA polymerase, but a binding site for the T4 transcriptional activator MotA replaces the sigma(70)-35 consensus. E. coli RNA polymerase transcribes from middle promoters with or without the activator. In contrast, transcription by T4-modified E. coli RNA polymerase, which is present during T4 infection, requires MotA. We show that transcription by unmodified polymerase from the T4 middle promoter P-uvsX is independent of the specific sequences within the -35 region, and the DNase I footprint obtained with unmodified polymerase and P-uvsX resembles those seen previously with E. coli ''extended -10'' promoters. In contrast, although T4-modified polymerase alone binds P-uvsX, promoter unwinding and detection of a DNase I footprint requires MotA. This footprint is significantly different from that obtained with unmodified polymerase, starting upstream of around position -20. Previous work has indicated that the T4 AsiA protein, which binds tightly to sigma(70), is the phage modification required for MotA activation. We show that in the presence of AsiA, MotA, and otherwise unmodified polymerase, DNase I protection of P-uvsX is now similar to that obtained with the fully modified polymerase and MotA up to around position -40. However, protection upstream of -40 is still similar to that seen with unmodified polymerase. Our results support the idea that MotA-dependent activation requires AsiA binding to sigma(70) to achieve specific protein-DNA contacts within the -20 to -40 region of a middle promoter. (C) 1996 Academic Press Limited