Mechanism of Action of the Escherichia coli Phage Shock Protein PspA in Repression of the AAA Family Transcription Factor PspF

Elderkin, Sarah; Jones, Susan; Schumacher, Julia; Studholme, David J.; Buck, Martin

doi:10.1016/s0022-2836(02)00404-7

Cited by 96 publications

(132 citation statements)

References 33 publications

Supporting

Mentioning

130

Contrasting

Order By: Relevance

“…Preparation of the PspA Complex-Plasmids, DNA manipulations, protein purification, and related analyses by SDS-PAGE and Western blotting have been described previously (12). Modification of the PspA purification protocol included the omission of 1.1% CHAPS from fractions prior to dialysis.…”

Section: Methodsmentioning

confidence: 99%

“…Samples were incubated for 10 min at 30°C and analyzed by 4.5% non-denaturing polyacrylamide gel electrophoresis (25 mM Tris-HCl, pH 8, and 200 mM glycine at 4°C). isomerization assays were conducted in 10-l reaction volumes as described previously (12). Essentially, STA buffer containing 16 nM 32 P end-labeled heteroduplex DNA (mismatched at Ϫ12/Ϫ11, consisting of the Ϫ60 to 28 Sinorhizobium meliloti nifH promoter), 1 mM 54 , 4 mM dGTP, and1 M PspF-(1-275) was pre-incubated with either storage buffer or increasing concentration of PspA (0.5, 1, and 2.5 M) in 4 l of TGED (100 mM NaCl, 10 mM Tris-HCl, pH 8, 0.1 mM EDTA, 1 mM dithiothreitol, and 50% glycerol) for 10 min at 30°C.…”

Section: Methodsmentioning

confidence: 99%

“…PspF is able to bind and hydrolyze ATP by using the derived energy to remodel the 54 -RNA polymerase. The PspA protein forms a complex with PspF, thereby inhibiting its function by stopping its ATPase activity, either by preventing nucleotide access to the active site or by inhibiting formation of the correct oligomeric form (12). PspA has recently been classified as an "adaptor" protein working with PspF (12)(13)(14).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Organization of the AAA+ Adaptor Protein PspA Is an Oligomeric Ring

Hankamer

Elderkin

Buck

et al. 2004

Journal of Biological Chemistry

Self Cite

114

View full text Add to dashboard Cite

The 25.3 kDa "adaptor" protein, PspA (phage shock protein A), is found in the cytoplasm and in association with the inner membrane of certain bacteria. PspA plays critical roles in negatively regulating the phage shock response and maintaining membrane integrity, especially during the export of proteins such as virulence factors. Homologues of PspA function exist for thylakoid biogenesis. Here we report the first three-dimensional reconstruction of a PspA assembly from Escherichia coli, visualized by electron microscopy and single particle analysis to a resolution of 30 Å. The assembly forms a 9-fold rotationally symmetric ring with an outer diameter of 200 Å, an inner diameter of 95 Å, and a height of ϳ85 Å. The molecular mass of the complex was calculated to be 1023 kDa by size exclusion chromatography, suggesting that each of the nine domains is likely to be composed of four PspA subunits. The functional implications of this PspA structure are discussed in terms of its interaction with the protein export machinery of the bacterial cell and its AAA ؉ protein partner, PspF.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Organization of the AAA+ Adaptor Protein PspA Is an Oligomeric Ring

Hankamer

Elderkin

Buck

et al. 2004

Journal of Biological Chemistry

Self Cite

114

View full text Add to dashboard Cite

show abstract

“…PspF, the activator of the pspA-E operon, occurs in cells at low concentrations and is constitutively active. Modulation of its activity, unlike that of NtrC, depends on protein regulators: PspA (negative) or PspB and PspC (positive) (Shingler, 1996;Elderkin et al, 2002;Hankamer et al, 2004). PspF responds to heat shock and ethanol treatment, but the strongest reaction is evoked by bacterial membrane impairments coincident with the dissipation of charge as by the gene IV protein of filamentous phages (e.g.…”

Section: Introductionmentioning

confidence: 99%

A σ 54-dependent promoter in the regulatory region of the Escherichia coli rpoH gene

et al. 2007

View full text Add to dashboard Cite

The Escherichia coli rpoH gene is transcribed from four known and differently regulated promoters: P1, P3, P4 and P5. This study demonstrates that the conserved consensus sequence of the s 54 promoter in the regulatory region of the rpoH gene, described previously, is a functional promoter, P6. The evidence for this conclusion is: (i) the specific binding of the s 54 -RNAP holoenzyme to P6, (ii) the location of the transcription start site at the predicted position (C, 30 nt upstream of ATG) and (iii) the dependence of transcription on s 54 and on an ATP-dependent activator. Nitrogen starvation, heat shock, ethanol and CCCP treatment did not activate transcription from P6 under the conditions examined. Two activators of s 54 promoters, PspF and NtrC, were tested but neither of them acted specifically. Therefore, PspFDHTH, a derivative of PspF, devoid of DNA binding capability but retaining its ATPase activity, was used for transcription in vitro, taking advantage of the relaxed specificity of ATP-dependent activators acting in solution. In experiments in vivo overexpression of PspFDHTH from a plasmid was employed. Thus, the s 54 -dependent transcription capability of the P6 promoter was demonstrated both in vivo and in vitro, although the specific conditions inducing initiation of the transcription remain to be elucidated. The results clearly indicate that the closed s 54 -RNAP-promoter initiation complex was formed in vitro and in vivo and needed only an ATP-dependent activator to start transcription.

show abstract

“…In order to better understand the intimate interactions made between the EBP and s 54 -RNAP, Bordes and colleagues (1) conducted protein fragmentation studies with the E. coli EBP phage shock protein F (PspF), a transcription regulator involved in stress response. (21,22) The fragmentation approach was adopted to overcome the problem of overlapping functionality associated with full-length protein-the activities of the full-length protein are interlinked. Therefore the fragmentation approach can be used to isolate or uncouple individual functions within specific sequence motifs.…”

Section: Introductionmentioning

confidence: 99%

Investigating protein–protein interfaces in bacterial transcription complexes: a fragmentation approach

Burrows

2003

BioEssays

View full text Add to dashboard Cite

SummaryTranscription initiation by s 54 -RNA polymerase (RNAP) relies explicitly on a transient interaction with a complex molecular machine belonging to the AAAþ (ATPases associated with various cellular activities) superfamily. Members of the AAAþ superfamily convert chemical energy derived from NTP hydrolysis to a mechanical force used to remodel their target substrate. Recently Bordes and colleagues, (1) using a protein fragmentation approach, identified a unique sequence within s 54 -dependent transcriptional activators that constitutes a s 54 -binding interface. This interface is not static, but subject to nucleotide-dependent movement which may represent a common mechanism for controlling output that has been adopted by other AAAþ proteins. IntroductionDefining the types of strategy and the mechanisms used to control gene expression is important for a full understanding of how the genetic information stored within DNA can be unlocked. Elaborate signal pathways exist to ensure that genes are only expressed when required, thus enabling flexible adaptation in response to changing environmental conditions. At the heart of these pathways lie transcription factors, which regulate the activity of DNA-dependent RNA polymerase (RNAP), the central enzyme in gene expression. Bacterial RNAP is a multisubunit enzyme with subunit composition a 2 bb 0 o; In this 'core' form, the RNAP is catalytically competent but incapable of locating specific DNA target sequences,

show abstract

Mechanism of Action of the Escherichia coli Phage Shock Protein PspA in Repression of the AAA Family Transcription Factor PspF

Cited by 96 publications

References 33 publications

Organization of the AAA+ Adaptor Protein PspA Is an Oligomeric Ring

Organization of the AAA+ Adaptor Protein PspA Is an Oligomeric Ring

A σ 54-dependent promoter in the regulatory region of the Escherichia coli rpoH gene

Investigating protein–protein interfaces in bacterial transcription complexes: a fragmentation approach

Contact Info

Product

Resources

About