Inhibition of Escherichia coli RNA polymerase by bacteriophage T4 AsiA

The 10 kDa bacteriophage T4 antisigma protein AsiA binds the Escherichia coli RNA polymerase promoter specificity subunit, σ⁷⁰, with high affinity and inhibits its transcription activity. AsiA binds to σ⁷⁰ primarily through an interaction with conserved region 4.2, which has also been implicated in sequence-specific recognition of the -35 consensus promoter element. Here we show that AsiA forms a stable ternary complex with core RNA polymerase (RNAP) and σ⁷⁰ and thus does not inhibit σ⁷⁰ activity by preventing its binding to core RNAP. We investigated the effect of AsiA on open promoter complex formation and abortive initiation at two -10/-35 type promoters and two 'extended -10' promoters. Our results indicate that the binding of AsiA to σ⁷⁰ and the interaction of σ⁷⁰ region 4.2 with the -35 consensus promoter element of -10/-35 promoters is mutually exclusive. In contrast, AsiA has much less effect on open promoter complex formation and abortive initiation from extended -10 promoters, which lack a -35 consensus element and do not require σ⁷⁰ conserved region 4.2. From these results we conclude that T4 AsiA inhibits E. coli RNAP σ⁷⁰ holoenzyme transcription at -10/ -35 promoters by interfering with the required interaction between σ⁷⁰ conserved region 4.2 and the -35 consensus promoter element.