The bacteriophage T4 AsiA protein contacts the β-flap domain of RNA polymerase

Abstract

To initiate transcription from specific promoters, the bacterial RNA polymerase (RNAP) core enzyme must associate with the initiation factor sigma, which contains determinants that allow sequence-specific interactions with promoter DNA. Most bacteria contain several sigma factors, each of which directs recognition of a distinct set of promoters. A large and diverse family of proteins known as "anti-sigma factors" regulates promoter utilization by targeting specific sigma factors. The founding member of this family is the AsiA protein of bacteriophage T4. AsiA specifically targets the primary sigma factor in Escherichia coli, sigma(70), and inhibits transcription from the major class of sigma(70)-dependent promoters. AsiA-dependent transcription inhibition has been attributed to a well-documented interaction between AsiA and conserved region 4 of sigma(70). Here, we establish that efficient AsiA-dependent transcription inhibition also requires direct protein-protein contact between AsiA and the RNAP core. In particular, we demonstrate that AsiA contacts the flap domain of the RNAP beta-subunit (the beta-flap). Our findings support the emerging view that the beta-flap is a target site for regulatory proteins that affect RNAP function during all stages of the transcription cycle.