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.
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