A point mutation at the junction of domain 2?3/2?4 of transcription factor σ⁷⁰ abrogates productive transcription and restores its expected mobility on a denaturing gel

Abstract

Region 2 of eubacterial s factors is highly conserved and the subdomain 2?4 is involved in -10 promote recognition. An evolutionary conserved "RpoD box" has been identified at the junction of subdomain 2?3/2?4 in class I and class II σ factors and there are two tryptophan residues at position 433 and 434 which can be used as intrinsic fluorescent markers to study their structure-function relationship. Site-directed mutagenesis of these two tryptophan residues has been carried out to generate three variants of σ⁷⁰ of Escherichia coli RNA polymerase. These are W433F, W433G and W434G. σ⁷⁰-W433F is found to be indistinguishable from the native σ factor by both structural and functional analysis. σ⁷⁰-W433G shows anomalous mobility on SDS-PAGE like the native σ factor, is σ -helical in conformation (50% helicity although found to be less active in total transcription when reconstituted with core RNA polymerase. Free σ⁷⁰-W434G, unlike the native σ factor, shows the expected mobility of a 70 kDa protein on SDS-PAGE and has 20% helicity. Time-resolved fluorescence analysis indicates that free σ⁷⁰-W434G has DNA binding ability, and displays a normal abortive initiation reaction but a decreased level of productive transcription after reconstitution with core RNA polymerase. A model is proposed in which tryptophan at position 434 interacts with the hydrophobic 1?1 domain of σ⁷⁰ giving rise to the stability of the protein under denaturing conditions.