The sigma 54 DNA‐binding domain includes a determinant of enhancer responsiveness

Chaney, Matthew; Buck, Martin

doi:10.1046/j.1365-2958.1999.01566.x

Cited by 51 publications

(35 citation statements)

References 38 publications

(93 reference statements)

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“…For residues 33-38 and 45-47, binding activity correlates with the critical role of these patches in activated transcription (Syed and Gralla 1998;Gallegos and Buck 2000). Importantly, two mutants in 54 (deletion 310-328 and R336A) that share with certain 54 Region I mutants and the Region I deletion form of 54 the property of activator-independent transcription in vitro (Chaney and Buck 1999;Chaney et al 2000), efficiently formed trapped complexes with PspF⌬HTH (data not shown). This further supports the argument that 54 Region I may directly interact with PspF⌬HTH.…”

Section: Role Of Activator Self-association In Binding 54mentioning

confidence: 89%

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP–aluminum fluoride: insights into activator mechanochemical action

Chaney¹,

Grande²,

et al. 2001

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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).

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Section: Role Of Activator Self-association In Binding 54mentioning

confidence: 89%

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP–aluminum fluoride: insights into activator mechanochemical action

Chaney¹,

Grande²,

et al. 2001

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“…The requirement for an extrinsic activator and ATP hydrolysis makes the mechanism of σ N -mediated transcription initiation strikingly different from the predominant σ 70 -family mechanism (1, 7). Mutants of σ N that allow open complex formation and transcription initiation without activator intervention (so-called bypass mutants) have been described (32)(33)(34)(35)(36)(37). RI of σ N has been proposed to contact activator directly (38, 39) and plays a critical role in mediating the response of the Eσ N /promoter complex to activator, and most bypass mutants comprise deletions or amino acid substitutions within RI (32, 34, 35, 40).…”

Section: Significancementioning

confidence: 99%

“…RI of σ N has been proposed to contact activator directly (38, 39) and plays a critical role in mediating the response of the Eσ N /promoter complex to activator, and most bypass mutants comprise deletions or amino acid substitutions within RI (32, 34, 35, 40). The only described bypass mutants that are not within RI are two absolutely conserved residues within the −12BD, corresponding to Kpn σ N R336 and K388 (Aae σ N R264 and K315) (36,37). Neither of these residues plays a role in DNA binding in the Aae ΔNσ N /DNA structure (Fig.…”

Section: Significancementioning

confidence: 99%

Crystal structure of Aquifex aeolicus σ ^N bound to promoter DNA and the structure of σ ^N -holoenzyme

Campbell

Kamath

Rajashankar

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The bacterial σ factors confer promoter specificity to the RNA polymerase (RNAP). One alternative σ factor, σ N , is unique in its structure and functional mechanism, forming transcriptionally inactive promoter complexes that require activation by specialized AAA + ATPases. We report a 3.4-Å resolution X-ray crystal structure of a σ N fragment in complex with its cognate promoter DNA, revealing the molecular details of promoter recognition by σ N . The structure allowed us to build and refine an improved σ N -holoenzyme model based on previously published 3.8-Å resolution X-ray data. The improved σ N -holoenzyme model reveals a conserved interdomain interface within σ N that, when disrupted by mutations, leads to transcription activity without activator intervention (so-called bypass mutants). Thus, the structure and stability of this interdomain interface are crucial for the role of σ N in blocking transcription activity and in maintaining the activator sensitivity of σ N .RNA polymerase | σ54 | σN | transcription | X-ray crystallography

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“…To further explore the activator ATPase-dependent actions giving rise to spRNA synthesis, we examined the properties of a σ 54 variant, R336A (in the context of Eσ 54 ), which is capable of transcribing in vitro in the absence of an activator ATPase and its nucleotide-dependent action (termed activator bypass transcription) (41)(42)(43). Strikingly, the R336A variant (which was specifically chosen because the activator ATPase-interacting domain remained intact) showed the exact same template specificities evident in ADP-AlF-dependent spRNA synthesis-where distinctions between dATP-and ADPAlF-dependent spRNA synthesis were apparent (Fig.…”

mentioning

confidence: 99%

A prehydrolysis state of an AAA+ ATPase supports transcription activation of an enhancer-dependent RNA polymerase

Burrows

Joly

Buck

2010

Proc. Natl. Acad. Sci. U.S.A.

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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...

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The sigma 54 DNA‐binding domain includes a determinant of enhancer responsiveness

Cited by 51 publications

References 38 publications

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP–aluminum fluoride: insights into activator mechanochemical action

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP–aluminum fluoride: insights into activator mechanochemical action

Crystal structure of Aquifex aeolicus σ ^N bound to promoter DNA and the structure of σ ^N -holoenzyme

A prehydrolysis state of an AAA+ ATPase supports transcription activation of an enhancer-dependent RNA polymerase

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The sigma 54 DNA‐binding domain includes a determinant of enhancer responsiveness

Cited by 51 publications

References 38 publications

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP–aluminum fluoride: insights into activator mechanochemical action

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP–aluminum fluoride: insights into activator mechanochemical action

Crystal structure of Aquifex aeolicus σ N bound to promoter DNA and the structure of σ N -holoenzyme

A prehydrolysis state of an AAA+ ATPase supports transcription activation of an enhancer-dependent RNA polymerase

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Crystal structure of Aquifex aeolicus σ ^N bound to promoter DNA and the structure of σ ^N -holoenzyme