Both regions 4.1 and 4.2 of E. coli σ⁷⁰ are together required for binding to bacteriophage T4 AsiA in vivo

Abstract

The T4 AsiA is an anti-sigma factor encoded by one of the early genes of Bacteriophage T4. It has been shown that AsiA inhibits transcription from promoters containing - 10 and - 35 consensus sequence by binding to σ ⁷⁰ of E. coli. Binding of AsiA to σ⁷⁰ in vivo, in E. coli, leads to inhibition of transcription of essential genes resulting in killing of the organism. By using various in vitro methods, the region of σ⁷⁰ binding to AsiA have been mapped to domain 4.2. Additionally, mutational analysis of σ⁷⁰ has also identified amino acid residues in domain 4.1 which are critical for interaction with AsiA. Based on NMR studies it has been suggested that either of these regions can bind to AsiA, a conclusion which was supported by high degree of amino acid homology between domain 4.1 and 4.2. However, it is not clear whether under in vivo conditions, AsiA exerts its transcription inhibitory effect by binding to one of these regions or both the regions together. In order to understand the mechanism of AsiA mediated inhibition of E. coli transcription in vivo, in terms of specific binding requirements to region 4.1 and/or 4.2, we have studied the interaction of these sub-domains with AsiA by Yeast two hybrid system as well as by co-expressing and affinity purification of the interacting partners in vivo in E. coli. It was observed that minimum fragment of σ⁷⁰ showing observable binding to AsiA, must possess sub-domains 4.1 and 4.2 together. No binding could be detected in σ⁷⁰ fragments lacking a part of either domain 4.1 or 4.2, in any of the assays. This data was also supported by in vitro binding studies wherein only σ⁷⁰ fragments carrying both region 4.1 and 4.2 showed binding to AsiA. Co-expression of region 4.1 and 4.2 fragments together also did not show any interaction with AsiA. The results presented here suggest that binding of AsiA to σ⁷⁰, in vivo, requires the presence of both sub-domains of region 4 of σ⁷⁰.