Abstract
The sigma(54) promoter specificity factor is distinct from other bacterial RNA polymerase (RNAP) sigma factors in that it forms a transcriptionally silent closed complex upon promoter binding. Transcriptional activation occurs through a nucleotide-dependent isomerization of sigma(54), mediated via its interactions with an enhancer-binding activator protein that utilizes the energy released in ATP hydrolysis to effect structural changes in sigma(54) and core RNA polymerase. The organization of sigma(54)-promoter and sigma(54)-RNAP-promoter complexes was investigated by fluorescence resonance energy transfer assays using sigma(54) single cysteine-mutants labeled with an acceptor fluorophore and donor fluorophore-labeled DNA sequences containing mismatches that mimic nifH early- and late-melted promoters. The results show that sigma(54) undergoes spatial rearrangements of functionally important domains upon closed complex formation. sigma(54) and sigma(54)-RNAP promoter complexes reconstituted with the different mismatched DNA constructs were assayed by the addition of the activator phage shock protein F in the presence or absence of ATP and of non-hydrolysable analogues. Nucleotide-dependent alterations in fluorescence resonance energy transfer efficiencies identify different functional states of the activator-sigma(54)-RNAP-promoter complex that exist throughout the mechano-chemical transduction pathway of transcriptional activation, i.e. from closed to open promoter complexes. The results suggest that open complex formation only occurs efficiently on replacement of a repressive fork junction with down-stream melted DNA.
Highlights
Sequence-specific binding of the multisubunit bacterial core RNA polymerase (RNAP,2 ␣2Ј (E)) to promoter DNA is conferred by an additional subunit, the factor
The results show that 54 undergoes spatial rearrangements of functionally important domains upon closed complex formation. 54 and 54-RNAP promoter complexes reconstituted with the different mismatched DNA constructs were assayed by the addition of the activator phage shock protein F in the presence or absence of ATP and of non-hydrolysable analogues
Truncated versions of 54 lacking Region I (54⌬RI) can bind to core RNAP, forming E54⌬RI complexes capable of activator-independent transcription from supercoiled and so called late-melted promoters that are mismatched between positions Ϫ10 and Ϫ1, mimicking the conformation adopted by the promoter DNA in the open complex but not on DNA constructs mismatched between Ϫ12 and Ϫ11, which mimic the conformation adopted by the promoter in the closed complex
Summary
E 28 Ϯ 4 22 Ϯ 4 19 Ϯ 5 20 Ϯ 5 a Activation of -DNA and isomerized supershifted -DNA complexes (ss) in the presence of PspF1–275 and ATP. b Activated E represents complexes isomerized in the presence of PspF1–275 and ATP that remain after heparin challenge. Bound to ADP1⁄7AlFx [27, 28] Using these structural data, sitedirected PspF mutants were assayed for ATP binding and hydrolysis activity and the ability to trigger open complex formation. It appears that structural changes occurring in the nucleotide binding pockets formed by the PspF hexamer are communicated via relocation of a conserved loop motif to make stable contacts with 54 [27]. FRET assays of the interactions of PspF1– 275 with 54 and E54 promoter complexes (formed on the early- or late-melted fragments) in the presence or absence of hydrolysable and non-hydrolysable nucleotides provide novel insights into the structural alterations occurring during formation of the open complex
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
