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A perspective on the enhancer dependent bacterial RNA polymerase.

Zhang N, Buck M - Biomolecules (2015)

Bottom Line: Here we review recent findings and offer a perspective on how the major variant RNA polymerase of bacteria, which contains the sigma54 factor, functions for regulated gene expression.We consider what gaps exist in our understanding of its genetic, biochemical and biophysical functioning and how they might be addressed.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, UK. nan.zhang@imperial.ac.uk.

ABSTRACT
Here we review recent findings and offer a perspective on how the major variant RNA polymerase of bacteria, which contains the sigma54 factor, functions for regulated gene expression. We consider what gaps exist in our understanding of its genetic, biochemical and biophysical functioning and how they might be addressed.

Show MeSH

Related in: MedlinePlus

Domain architecture and the proposed mechanism of activation. (A) Cryo-EM reconstitution of the Eσ54-PspF-ADP-AlFx with the promoter DNA modelled in [21]; (B) Domain movements of σ54 Regions I and III during the transcription activation cycle (modified from [24]). The white stars depict fluorophores used in the smFRET experiments.
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biomolecules-05-01012-f002: Domain architecture and the proposed mechanism of activation. (A) Cryo-EM reconstitution of the Eσ54-PspF-ADP-AlFx with the promoter DNA modelled in [21]; (B) Domain movements of σ54 Regions I and III during the transcription activation cycle (modified from [24]). The white stars depict fluorophores used in the smFRET experiments.

Mentions: Sigma54 activators (such as NtrC and PspF) belonging to the AAA+ protein family assemble into hexamers and fuel the rearrangement of RPC to RPO. Cryo-EM reconstitutions carried out by the Zhang lab revealed up to three sigma54 activators within a hexamer could directly contact the RPC structure for isomerisation [21]. These contacts were made asymmetrically via the GAFTGA loop one motifs to sigma54 Region I and the upstream −30 promoter region [22], and were possibly accompanied by the splitting of the hexameric ATPase ring in order to exert directional forces [23]. A bridging density within the holoenzyme was observed to physically block the DNA loading channel formed between the β and β’ subunits prior to activation (Figure 2A). This density was assigned to a part of sigma54 Region I and it relocated downstream towards the +1 site in the intermediate complex (RPI) when the ADP-AlFx hydrolysis analogue was added. The DNA melting site was misaligned with the DNA loading site in the RPI in the proposed model, which could constitute an ancillary inhibitory mechanism. The Stockley and Tuma labs further addressed sigma54 domain movements in relation to promoter DNA and ATP hydrolysis by smFRET analysis (Figure 2B, [24]). Sigma54 Region I moved by approximately 9 Å towards the leading edge of the −10 to −1 transcription bubble in the RPI when activated with ADP-AlFx. This downstream movement upon activation fully agreed with the Cryo-EM observations and potentially correlated with blockage removal by the “power stroke” action of activator ATPases. Once ATP was fully hydrolysed, Region I would retract slightly upstream, possibly to accompany the DNA loading event. In contrast, Region III remained rather static with respect to the −24 promoter sequence along the activation pathway for making RPO from RPC.


A perspective on the enhancer dependent bacterial RNA polymerase.

Zhang N, Buck M - Biomolecules (2015)

Domain architecture and the proposed mechanism of activation. (A) Cryo-EM reconstitution of the Eσ54-PspF-ADP-AlFx with the promoter DNA modelled in [21]; (B) Domain movements of σ54 Regions I and III during the transcription activation cycle (modified from [24]). The white stars depict fluorophores used in the smFRET experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496707&req=5

biomolecules-05-01012-f002: Domain architecture and the proposed mechanism of activation. (A) Cryo-EM reconstitution of the Eσ54-PspF-ADP-AlFx with the promoter DNA modelled in [21]; (B) Domain movements of σ54 Regions I and III during the transcription activation cycle (modified from [24]). The white stars depict fluorophores used in the smFRET experiments.
Mentions: Sigma54 activators (such as NtrC and PspF) belonging to the AAA+ protein family assemble into hexamers and fuel the rearrangement of RPC to RPO. Cryo-EM reconstitutions carried out by the Zhang lab revealed up to three sigma54 activators within a hexamer could directly contact the RPC structure for isomerisation [21]. These contacts were made asymmetrically via the GAFTGA loop one motifs to sigma54 Region I and the upstream −30 promoter region [22], and were possibly accompanied by the splitting of the hexameric ATPase ring in order to exert directional forces [23]. A bridging density within the holoenzyme was observed to physically block the DNA loading channel formed between the β and β’ subunits prior to activation (Figure 2A). This density was assigned to a part of sigma54 Region I and it relocated downstream towards the +1 site in the intermediate complex (RPI) when the ADP-AlFx hydrolysis analogue was added. The DNA melting site was misaligned with the DNA loading site in the RPI in the proposed model, which could constitute an ancillary inhibitory mechanism. The Stockley and Tuma labs further addressed sigma54 domain movements in relation to promoter DNA and ATP hydrolysis by smFRET analysis (Figure 2B, [24]). Sigma54 Region I moved by approximately 9 Å towards the leading edge of the −10 to −1 transcription bubble in the RPI when activated with ADP-AlFx. This downstream movement upon activation fully agreed with the Cryo-EM observations and potentially correlated with blockage removal by the “power stroke” action of activator ATPases. Once ATP was fully hydrolysed, Region I would retract slightly upstream, possibly to accompany the DNA loading event. In contrast, Region III remained rather static with respect to the −24 promoter sequence along the activation pathway for making RPO from RPC.

Bottom Line: Here we review recent findings and offer a perspective on how the major variant RNA polymerase of bacteria, which contains the sigma54 factor, functions for regulated gene expression.We consider what gaps exist in our understanding of its genetic, biochemical and biophysical functioning and how they might be addressed.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, UK. nan.zhang@imperial.ac.uk.

ABSTRACT
Here we review recent findings and offer a perspective on how the major variant RNA polymerase of bacteria, which contains the sigma54 factor, functions for regulated gene expression. We consider what gaps exist in our understanding of its genetic, biochemical and biophysical functioning and how they might be addressed.

Show MeSH
Related in: MedlinePlus