Normal cell development requires the interaction of RNA polymerase with many factors that regulate transcriptional activity. We study transcription from bacteriophage T4 middle promoters in which the initiation of transcription by E. coli RNA polymerase requires the phage-encoded transcriptional activator, MotA protein, and the T4 co-activator, AsiA protein. This system is a simple model for examining how factors can change the specificity of a polymerase. E. coli RNA polymerase holoenzyme is composed of a core of 4 subunits plus a sigma factor that recognizes specific promoter sequences. In the past year, we have been investigating how mutations within the 613 amino acids of sigma-70, the major sigma subunit in E. coli, affect transcription. Previous work has shown that the far C-terminal domain of sigma-70 (region 4) is required to interact both with the -35 region of bacterial promoters and with particular bacterial activators. We have demonstrated that sigma-70 protein lacking this domain cannot bind to MotA or to AsiA and does not support MotA/AsiA activated transcription. These results suggest that MotA/AsiA activation of T4 middle promoters is achieved through the interaction of the phage proteins with region 4 of sigma-70. A previous study of a set of seventeen sigma-70 missense mutants revealed that specific C-terminal amino acids are required for interacting with particular bacterial activators. However, we have found that each of these mutants supports middle transcription in vivo, suggesting that the MotA/AsiA interaction with sigma-70 differs from that of activators in the bacterial systems. Although sigma-70 recognizes promoter DNA when it is present in polymerase holoenzyme, sigma-70 by itself does not bind DNA. It has been shown that the first 100 amino acids of sigma-70 region 1.1) are responsible for inhibiting DNA binding by sigma-70 alone. We have investigated the role of region 1.1 when sigma- 70 is present in holoenzyme. We have found that the ability of holoenzyme to transcribe from specific promoters is modulated by region 1.1. A holoenzyme with a sigma-70 that lacks region 1.1 is less active than holoenzyme with a full length sigma-70 in transcribing from strong promoters. However, it more readily uses a weak promoter, designated Pminor. We have shown that holoenzyme that lacks region 1.1 is more active at Pminor because it rapidly forms stable complexes with this promoter while holoenzyme with a full length sigma-70 does not. We have also found that the T4 coactivator AsiA binds more readily to sigma-70 that lacks region 1.1 than it binds to full length sigma-70. Our results suggest that the N-terminal region of sigma-70 (region 1.1) modulates the accessibility of the C- terminal region of sigma-70 (region 4) both for promoter DNA and for regulatory factors.
