Matthew Chaney

Mechanochemical ATPases and transcriptional activation

Transcriptional activator proteins that act upon the σ54‐containing form of the bacterial RNA polymerase belong to the extensive AAA+ superfamily of ATPases, members of which are found in all three kingdoms of life and function in diverse cellular processes, often via chaperone‐like activities. Formation and collapse of the transition state of ATP for hydrolysis appears to engender the interaction of the activator proteins with σ54 and leads to the protein structural transitions needed for RNA polymerase to isomerize and engage with the DNA template strand. The common oligomeric structures of AAA+ proteins and the crea‐tion of the active site for ATP hydrolysis between protomers suggest that the critical changes in protomer structure required for productive interactions with σ54‐holoenzyme occur as a consequence of sensing the state of the γ‐phosphate of ATP. Depending upon the form of nucleotide bound, different functional states of the activator are created that have distinct substrate and chaperone‐like binding activ‐ities. In particular, interprotomer ATP interactions rely upon the use of an arginine finger, a situation reminiscent of GTPase‐activating proteins.

The ATP hydrolyzing transcription activator phage shock protein F of Escherichia coli: Identifying a surface that binds σ54

Members of the protein family called ATPases associated with various cellular activities (AAA+) play a crucial role in transforming chemical energy into biological events. AAA+ proteins are complex molecular machines and typically form ring-shaped oligomeric complexes that are crucial for ATPase activity and mechanism of action. The Escherichia coli transcription activator phage shock protein F (PspF) is an AAA+ mechanochemical enzyme that functions to sense and relay the energy derived from nucleoside triphosphate hydrolysis to catalyze transcription by the σ54-RNA polymerase. Closed promoter complexes formed by the σ54-RNA polymerase are substrates for the action of PspF. By using a protein fragmentation approach, we identify here at least one σ54-binding surface in the PspF AAA+ domain. Results suggest that ATP hydrolysis by PspF is coupled to the exposure of at least one σ54-binding surface. This nucleotide hydrolysis-dependent presentation of a substrate binding surface can explain why complexes that form between σ54 and PspF are transient and could be part of a mechanism used generally by other AAA+ proteins to regulate activity.

THE SIGMA 54 DNA-BINDING DOMAIN INCLUDES A DETERMINANT OF ENHANCER RESPONSIVENESS

The bacterial σ54 protein associates with core RNA polymerase to form a holoenzyme that functions in enhancer‐dependent transcription. Isomerization of the σ54 polymerase and its engagement with melted DNA in open promoter complexes requires nucleotide hydrolysis by an enhancer‐binding activator. We show that a single amino acid substitution, RA336, in the Klebsiella pneumoniaeσ54 C‐terminal DNA‐binding domain allows the holoenzyme to isomerize, engage with stably melted DNA and to transcribe from transiently melting DNA without an activator. Activator responsiveness for the formation of stable open complexes remained intact. The activator‐independent transcription phenotype of RA336 is shared with mutants in amino‐terminal Region I sequences. Thus, in σ54, two distinct domains function for enhancer responsiveness. A σ54‐DNA contact mediated by R336 appears to be part of a network of interactions necessary for maintaining the transcriptionally inactive state of the holoenzyme. We suggest activator functions to change these interactions and facilitate open complex formation through promoting polymerase isomerization.

Communication between Eσ54, promoter DNA and the conserved threonine residue in the GAFTGA motif of the PspF σ54-dependent activator during transcription activation

Conversion of Eσ54 closed promoter complexes to open promoter complexes requires specialized activators which are members of the AAA (ATPases Associated with various cellular Activities) protein family. The ATP binding and hydrolysis activity of Eσ54 activators is used in an energy coupling reaction to remodel the Eσ54 closed promoter complex and to overcome the σ54‐imposed block on open complex formation. The remodelling target for the AAA activator within the Eσ54 closed complex includes a complex interface contributed to by Region I of σ54, core RNA polymerase and a promoter DNA fork junction structure, comprising the Eσ54 regulatory centre. One σ54 binding surface on Eσ54 activators is a conserved sequence known as the GAFTGA motif. Here, we present a detailed characterization of the interaction between Region I of σ54 and the Escherichia coli AAA σ54 activator Phage shock protein F. Using Eσ54 promoter complexes that mimic different conformations adopted by the DNA during open complex formation, we investigated the contribution of the conserved threonine residue in the GAFTGA motif to transcription activation. Our results suggest that the organization of the Eσ54 regulatory centre, and in particular the conformation adopted by the σ54 Region I and the DNA fork junction structure during open complex formation, is communicated to the AAA activator via the conserved T residue of the GAFTGA motif.

Involvement of the sigmaN DNA-binding domain in open complex formation.

σN (σ54) RNA polymerase holoenzyme closed complexes isomerize to open complexes in a reaction requiring nucleoside triphosphate hydrolysis by enhancer binding activator proteins. Here, we characterize Klebsiella pneumoniaeσN mutants, altered in the carboxy DNA‐binding domain (F354A/F355A, F402A, F403A and F402A/F403A), that fail in activator‐dependent transcription. The mutant holoenzymes have altered activator‐dependent interactions with promoter sequences that normally become melted. Activator‐dependent stable complexes accumulated slowly in vitro (F402A) and to a reduced final level (F403A, F402A/F403A, F354A/F355A). Similar results were obtained in an assay of activator‐independent stable complex formation. Premelted templates did not rescue the mutants for stable preinitiation complex formation but did for deleted region I σN, suggesting different defects. The DNA‐binding domain substitutions are within σN sequences previously shown to be buried upon formation of the wild‐type holoenzyme or closed complex, suggesting that, in the mutants, alteration of the σN–core and σN–DNA interfaces has occurred to change holoenzyme activity. Core‐binding assays with the mutant sigmas support this view. Interestingly, an internal deletion form of σN lacking the major core binding determinant was able to assemble into holoenzyme and, although unable to support activator‐dependent transcription, formed a stable activator‐independent holoenzyme promoter complex on premelted DNA templates.

Sequences within the DNA Cross-linking Patch of ς54Involved in Promoter Recognition, ς Isomerization, and Open Complex Formation

The bacterial RNA polymerase holoenzyme containing the ς54 subunit functions in enhancer-dependent transcription. Mutagenesis has been used to probe the function of a sequence in the ς54 DNA binding domain that includes residues that cross-link to promoter DNA. Several activities of the ς and holoenzyme are shown to depend on the cross-linking patch. The patch contributes to promoter binding by ς54, and holoenzyme and is involved in activator-dependent ς isomerization. As part of the ς54-holoenzyme, some residues in the patch limit basal transcription. Other cross-linking patch sequences appear to limit activator-dependent open complex formation. Deletion of 19 residues adjacent to the cross-linking patch resulted in a holoenzyme unable to respond to activator but capable of activator-independent (bypass) transcription in vitro. Overall results are consistent with the cross-linking patch directing interactions to the −12 promoter region to set basal and activated levels of transcription.