The bacterial AAA+ enhancer-binding proteins (EBPs) HrpR and HrpS (HrpRS) of Pseudomonas syringae (Ps) activate σ54-dependent transcription at the hrpL promoter; triggering type-three secretion system-mediated pathogenicity. In contrast with singly acting EBPs, the evolution of the strictly co-operative HrpRS pair raises questions of potential benefits and mechanistic differences this transcription control system offers. Here, we show distinct properties of HrpR and HrpS variants, indicating functional specialization of these non-redundant, tandemly arranged paralogues. Activities of HrpR, HrpS and their control proteins HrpV and HrpG from Ps pv. tomato DC3000 in vitro establish that HrpRS forms a transcriptionally active hetero-hexamer, that there is a direct negative regulatory role for HrpV through specific binding to HrpS and that HrpG suppresses HrpV. The distinct HrpR and HrpS functionalities suggest how partial paralogue degeneration has potentially led to a novel control mechanism for EBPs and indicate subunit-specific roles for EBPs in σ54-RNA polymerase activation.

SummaryBacterial sigma (s) factors confer gene specificity upon the RNA polymerase, the central enzyme that catalyses gene transcription. The binding of the alternative s factor s 54 confers upon the RNA polymerase special functional and regulatory properties, making it suited for control of several major adaptive responses. Here, we summarize our current understanding of the interactions the s 54 factor makes with the bacterial transcription machinery.

SummaryTranscription initiation involves the conversion from closed promoter complexes, comprising RNA polymerase (RNAP) and double-stranded promoter DNA, to open complexes, in which the enzyme is able to access the DNA template in a single-stranded form. The complex between bacterial RNAP and its major variant sigma factor σ54 remains as a closed complex until ATP hydrolysis-dependent remodeling by activator proteins occurs. This remodeling facilitates DNA melting and allows the transition to the open complex. Here we present cryoelectron microscopy reconstructions of bacterial RNAP in complex with σ54 alone, and of RNAP-σ54 with an AAA+ activator. Together with photo-crosslinking data that establish the location of promoter DNA within the complexes, we explain why the RNAP-σ54 closed complex is unable to access the DNA template and propose how the structural changes induced by activator binding can initiate conformational changes that ultimately result in formation of the open complex.

To survive and colonise their various environments, including those used during infection, bacteria have developed a variety of adaptive systems. Amongst these is phage shock protein (Psp) response, which can be induced in Escherichia coli upon filamentous phage infection (specifically phage secretin pIV) and by other membrane-damaging agents. The E. coli Psp system comprises seven proteins, of which PspA is the central component. PspA is a bifunctional protein that is directly involved in (i) the negative regulation of the psp-specific transcriptional activator PspF and (ii) the maintenance of membrane integrity in a mechanism proposed to involve the formation of a 36-mer ring complex. Here we established that the PspA negative regulation of PspF ATPase activity is the result of a cooperative inhibition. We present biochemical evidence showing that an inhibitory PspA–PspF regulatory complex, which has significantly reduced PspF ATPase activity, is composed of around six PspF subunits and six PspA subunits, suggesting that PspA exists in at least two different oligomeric assemblies. We now establish that all four putative helical domains of PspA are critical for the formation of the 36-mer. In contrast, not all four helical domains are required for the formation of the inhibitory PspA–PspF complex. Since a range of initial PspF oligomeric states permit formation of the apparent PspA–PspF dodecameric assembly, we conclude that PspA and PspF demonstrate a strong propensity to self-assemble into a single defined heteromeric regulatory complex.

Sigma factors, the key regulatory components of the bacterial RNA polymerase (RNAP), direct promoter DNA binding and DNA melting. The r 54 -RNAP forms promoter complexes in which DNA melting is only triggered by an activator and ATP hydrolysis-driven reorganisation of an initial r 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 r 54 as a chemical nuclease we now show that the reorganisation of the initial r 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 r 54 -RNAP-promoter complex, DNA contacts across the 'to be melted' sequences are made by the r 54 subunit. Strikingly, the activator protein, but not core RNAP subunits, is close to these DNA sequences.

The 54 promoter specificity factor is distinct from 70 -type factors. The 54 -RNA polymerase binds to promoters with conserved sequence elements at ؊24 and ؊12 and utilizes specialized enhancer-binding activators to convert, through an ATP-dependent process, closed promoter complexes to open promoter complexes. The interface between 54 -RNA polymerase and promoter DNA is poorly characterized, contrasting with 70 . Here, 54 was modified with strategically positioned cleavage reagents to provide physical evidence that the highly conserved RpoN box motif of 54 is close to and may therefore interact with the consensus ؊24 promoter element. We show that the spatial relationship between the 54 -RNA polymerase and the ؊24 promoter element remains unchanged during closed to open complex conversion and transcription initiation but changes during the early elongation phase. In contrast, the spatial relationship between 54 -RNA polymerase and the consensus ؊12 promoter element changes upon conversion of the closed promoter complex to an open one. We provide evidence that some ؊12 promoter region-54 interactions are dependent upon either the core RNA polymerase or a fork junction DNA structure at the ؊12-position, indicating that DNA fork junctions can substitute for core RNAP. We also show the ␤-subunit flap domain contributes to different sets of -promoter DNA interactions at 54 -and 70 -dependent promoters.Transcription of DNA is a fundamental process needed for regulation of cellular adaptation and differentiation and is carried out by DNA-dependent RNA polymerases (RNAPs).

The phage-shock-protein (Psp) response maintains the proton-motive force (pmf) under extracytoplasmic stress conditions that impair the inner membrane (IM) in bacterial cells. In Escherichia coli transcription of the pspABCDE and pspG genes requires activation of σ54-RNA polymerase by the enhancer-binding protein PspF. A regulatory network comprising PspF–A–C–B–ArcB controls psp expression. One key regulatory point is the negative control of PspF imposed by its binding to PspA. It has been proposed that under stress conditions, the IM-bound sensors PspB and PspC receive and transduce the signal(s) to PspA via protein–protein interactions, resulting in the release of the PspA–PspF inhibitory complex and the consequent induction of psp. In this work we demonstrate that PspB self-associates and interacts with PspC via putative IM regions. We present evidence suggesting that PspC has two topologies and that conserved residue G48 and the putative leucine zipper motif are determinants required for PspA interaction and signal transduction upon stress. We also establish that PspC directly interacts with the effector PspG, and show that PspG self-associates. These results are discussed in the context of formation and function of the Psp regulatory complex.

M acromolecular motion occurs as a part of almost all major enzyme-driven processes and is essential for the biological function of enzymes and nucleic acids. Molecular machines and motors (often belonging to the P-loop NTPase superfamily) are capable of catalyzing a chemical reaction, capturing the free energy released from this reaction, and using this energy to perform biologically useful mechanical work. Molecular machines are present in all kingdoms of life and play essential roles in diverse biological processes ranging from muscle contraction to neuron development and mitosis to gene expression (1-3). The regulated conformational transformations they drive are achieved through distinctly different ATP-and ADP-bound functional states of such molecular machines and motors (4-6). However, which of the distinct nucleotide-bound states of the ATPases cause conformational change in their target substrates is often unknown (5).One important ATP-dependent gene regulation complex is that of the bacterial RNA polymerase (RNAP) containing the major variant σ 54 factor (Eσ 54 ). Transcription initiation is a multistep process (including the obligatory DNA-melting step) that is subject to tight regulation. RNAP binds to specific promoter DNA sequences, forming the closed complex, and then proceeds to melt the DNA sequences surrounding the transcription start site at +1 to yield an open complex (7-10). In the case of enhancer-dependent Eσ 54 , open complex formation strictly requires engagement of the closed complex with a specialized ATP-hydrolyzing enhancer binding transcriptional activator protein (a member of the AAA+ superfamily of P-loop ATPases) (11-18), resembling the transcription initiation process of eukaryotic RNAP II (19)(20)(21)(22). The promoter complexes formed with activated Eσ 54 support numerous crucial adaptive responses, including those required for bacterial pathogenesis and survival under extreme nutritional stress (23). One approach to investigate the action of the activator ATPase involves the use of metal fluoride analogs to capture the different nucleotidebound states associated with nucleotide binding and hydrolysis (5,24,25). These ATP analogs allow us to probe the differential contributions that the presence (and state) of the γ-β-phosphate bond (of ATP) makes to the functionality of ATPases by setting and restricting their nucleotide-bound state. One such nonhydrolyzable ATP analog is ADP-AlF, thought to represent ATP at the point of ATP hydrolysis (26). Using ADP-AlF and the catalytic AAA+ domain of the archetypal activator ATPase phage shock protein F (PspF 1-275 ), we can "trap" Eσ 54 and study the properties of this trapped complex (Eσ 54 -PspF 1-275 :ADP-AlF) (11,(24)(25)(26)(27)(28). Recent cryoelectron microscopy structural studies of this trapped complex demonstrated that activator binding to σ 54 reconfigures Eσ 54 to (i) help align the +1 promoter DNA with the RNAP active site and (ii) begin to make the DNA-binding cleft of RNAP available (11). However, the precise outcomes of t...