Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: Insights from single molecule studies

Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ⁷⁰ or σ⁵⁴, that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ⁵⁴ version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ⁷⁰ and σ⁵⁴, the domain movements of the latter have evolved to require an activator ATPase.