The kinetics of sigma subunit directed promoter recognition by E-coli RNA polymerase

Time-resolved laser UV irradiation and controlled proteolysis have been use d to study the sequential recognition of the Inc UV5 promoter by Escherichi a coli RNA polymerase. Local rearrangements in the DNA, the appearance of i ntimate protein-DNA contacts, and structural changes within the sigma subun it together provide specific signatures that define major species populated during this process. At 22 degrees C, a first closed complex is characteri sed by a transient conformational change in the sigma subunit and by a dist ortion in the -35 region. Subsequently, direct contacts at -34 and at posit ions -8, -5 and -3 on the non-template strand appear prior to DNA strand se paration. The contact in the -35 consensus region involves only the sigma s ubunit. This intermediate possesses different structural parameters from th at formed by quenching open complexes from 37 degrees C to 14 degrees C. Si gma thus appears as the principal partner acting during promoter recognitio n, a strongly coupled process involving two major intermediates only.