title: Single chain forms of the enhancer binding protein PspF provide insights into geometric requirements for gene activation.
abstract: Genetic information in the DNA is accessed by the molecular machine RNA polymerase following a highly conserved process, invariably involving the transition between double-stranded and single-stranded DNA states. In the case of the bacterial enhancer-dependent RNA polymerase (which is essential for adaptive responses and bacterial pathogenesis), the DNA melting event depends on specialized hexameric AAA+ ATPase activators. Involvement of such factors in transcription was demonstrated 25 years ago, but why these activators need to be hexameric, whether all the subunits operate identically, what is the contribution of each of the six subunits within the hexamer (structural, functional, or both), and how many active subunits are required for transcription activation remain open questions. Using engineered single-chain polypeptides covalently linking two or three subunits of the activator (allowing the subunit distribution within a hexamer to be fixed), we now show that (i) individual subunits have differential contributions to the activities of the oligomer and (ii) only a fraction of the subunits within the hexameric ATPase is directly required for gene activation. We establish that nucleotide-dependent coordination across three subunits of the hexameric bacterial enhancer binding proteins (bEBPs) is necessary for engagement and remodeling of the closed complex (RPc). Outcomes revealed features of bEBP, distinguishing their mode of action from fully processive AAA+ proteins or from simple bimodal switches. We now propose that the hexamer functions with asymmetric organization, potentially involving a split planar (open ring) or spiral character.
authors.affiliation: Division of Biology, Sir Alexander Fleming Building, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom. joly@ijm.univ-paris-diderot.fr
authors.forename: Nicolas
authors.initials: N
authors.lastname: Joly
authors.forename: Martin
authors.initials: M
authors.lastname: Buck
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008962:Models, Theoretical; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics / Q000502:physiology*
chemical_list: D029968:Escherichia coli Proteins; C098954:PspF protein, E coli; D015534:Trans-Activators; D012321:DNA-Directed RNA Polymerases; D000251:Adenosine Triphosphatases