At some bacterial promoters, a 5′-TG-3′ sequence element, located one base upstream of the −10 hexamer element, provides an essential motif necessary for transcription initiation. We have identified a mutant of the Escherichia coli RNA polymerase σ70 subunit that has an altered preference for base sequences in this ‘extended −10’ region. We show that this mutant σ70 subunit substantially increases transcription from promoters bearing 5′-TC-3′ or 5′-TT-3′ instead of a 5′-TG-3′ motif, located one base upstream of the −10 hexamer. The mutant results from a single base pair substitution in the rpoD gene that causes a Glu to Gly change at position 458 of σ70. This substitution identifies a functional region in σ70 that is immediately adjacent to the well-characterized region 2.4 (positions 434–453, previously shown to contact the −10 hexamer). From these results, we conclude that this region (which we name region 2.5) is involved in contacting the 5′-TG-3′ motif found at some bacterial promoters: thus, extended −10 regions are recognized by an extended region 2 of the RNA polymerase σ70 subunit.