Transcription by RNA polymerase (RNAP) is often regulated by interactions with control proteins to link specific gene expression to environmental signals and temporal cues. Often activators help recruit RNAP to promoters to increase initiation rates (Busby and Ebright 1999). In contrast, activity of the bacterial 54 containing RNAP holoenzyme is regulated at the DNA melting step (for review, see Buck et al. 2000). Hydrolysis of an NTP by an activator drives a change in configuration of the 54 -holoenzyme, converting the initial closed complex to an open complex to allow interaction with the template DNA for mRNA synthesis (Wedel and Kustu 1995). Preopening of DNA templates does not overcome the requirement for NTP hydrolysis by an activator to promote engagement of the holoenzyme with the melted DNA (Wedel and Kustu 1995;Cannon et al. 1999).The activators of 54 -holoenzyme are members of the large AAA+ protein family, which use ATP binding and hydrolysis to remodel their substrates (Neuwald et al. 1999;Cannon et al. 2000Cannon et al. , 2001. The greater part of the central domain of 54 activators corresponds to the AAA core structure, and includes ATP-binding and hydrolyzing determinants. The 54 protein is known to be the primary target for the NTPase of activators, but how activators use NTP binding and hydrolysis is not well understood (Cannon et al. 2000). Similarly, the nature of the interaction between 54 and the activator is not well described, but an interaction with 54 can be detected in the case of the DctD activator by protein cross-linking (Lee and Hoover 1995). Here we show that the use of ADP-aluminum fluoride, an analog of ATP that mimics ATP in the transition state for hydrolysis, allows formation of a stable complex among the activator PspF, the PspF and NifA central activating domains, and 54 . The binding assay was used to help define determinants in 54 and the activator needed for their interaction, and to show that binding can lead to an altered 54 -DNA footprint. The need for a transition-state analog of ATP for protein-protein binding is discussed in relation to the required ATPase activity of activators of 54 -dependent transcription. In particular, it seems that altered functional states of activators exist as ATP is hydrolyzed. This suggests a parallel to some switch and motor proteins that use nucleotide binding and hydrolysis to establish alternate functional states (Hirose and Amos 1999).

Summary 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.

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 54 -RNA polymerase. Closed promoter complexes formed by the 54 -RNA polymerase are substrates for the action of PspF. By using a protein fragmentation approach, we identify here at least one 54 -binding surface in the PspF AAA ؉ domain. Results suggest that ATP hydrolysis by PspF is coupled to the exposure of at least one 54 -binding surface. This nucleotide hydrolysis-dependent presentation of a substrate binding surface can explain why complexes that form between 54 and PspF are transient and could be part of a mechanism used generally by other AAA ؉ proteins to regulate activity.

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