Alteration in template recognition by Escherichia coli RNA polymerase lacking the w subunit: A mechanistic analysis through gel retardation and foot-printing studies

The w subunit of Escherichia coli RNA polymerase is a 91 amino acid polypeptide which co-purifies with the enzyme and is thought to help in maturing the rest of the enzyme to its full functionality. Purified w when added externally was found to inhibit general transcriptional activity of wless RNA polymerase as well as promoter-specific single-round transcriptional activity at all the promoters tested. In this study we have tried to analyse the observed inhibition of transcription using gel retardation assays and KMnO4 foot-printing. Further, through protein foot-printing we have attempted to identify alterations in the interaction of the w-less core enzyme with the s 70 subunit. Our results suggest that the w-less holoenzyme has lesser affinity towards the DNA template and external addition of w destabilizes the open complex for both the wild-type and w-less enzyme. The w-less core enzyme interacts with the s 70 subunit to expose the - 35 recognition domain (domain 4·2) unlike that observed in the wild-type interaction. Thus the absence of the w subunit leads to the formation of an enzyme which has altered DNA binding and s 70 binding properties. Circular dichroic measurements also indicate a major conformational alteration of both holo and core RNA polymerase in the presence and absence of the w subunit.