Reorganisation of an RNA polymerase-promoter DNA complex for DNA melting

Sigma factors, the key regulatory components of the bacterial RNA polymerase (RNAP), direct promoter DNA binding and DNA melting. The σ⁵⁴-RNAP forms promoter complexes in which DNA melting is only triggered by an activator and ATP hydrolysis-driven reorganisation of an initial σ⁵⁴-RNAP-promoter complex. We report that an initial bacterial RNAP-DNA complex can be reorganised by an activator to form an intermediate transcription initiation complex where full DNA melting has not yet occurred. Using σ⁵⁴ as a chemical nuclease we now show that the reorganisation of the initial σ⁵⁴-RNAP-promoter complex occurs upon interaction with the activator at the transition point of ATP hydrolysis. We demonstrate that this reorganisation event is an early step in the transcription initiation pathway that occurs independently of RNAP parts normally associated with stable DNA melting and open complex formation. Using photoreactive DNA probes, we provide evidence that within this reorganised σ⁵⁴-RNAP-promoter complex, DNA contacts across the 'to be melted' sequences are made by the σ⁵⁴ subunit. Strikingly, the activator protein, but not core RNAP subunits, is close to these DNA sequences.