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 sigma(54)-RNAP forms promoter complexes in which DNA melting is only triggered by an activator and ATP hydrolysis-driven reorganisation of an initial sigma(54)-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 sigma(54) as a chemical nuclease we now show that the reorganisation of the initial sigma(54)-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 sigma(54)-RNAP-promoter complex, DNA contacts across the 'to be melted' sequences are made by the sigma(54) subunit. Strikingly, the activator protein, but not core RNAP subunits, is close to these DNA sequences.
Department of Biological Sciences, Imperial College London, London SW7 2AZ, UK.
Patricia C
PC
Burrows
Konstantin
K
Severinov
Martin
M
Buck
Siva R
SR
Wigneshweraraj
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D017665:Hydroxyl Radical / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D009691:Nucleic Acid Denaturation; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000378:metabolism; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism*; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D021122:Protein Subunits; D012808:Sigma Factor; D017665:Hydroxyl Radical; D000244:Adenosine Diphosphate; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54