The transcription factor PspF, for Phage shock protein F, is a bacterial enhancer-binding protein required for σ54-dependent transcription activation [5, 6]. This regulator activates the transcription of the psp regulon, and it is negatively autoregulated and coordinately activated by transcription of the divergent operon psp [1, 4, 6, 7]. The integration host factor facilitates control of the psp regulon [2, 4, 8].
The psp regulon is defined like the phage shock protein system, which is involved in protecting the bacterium during infectious processes [4, 6]. The synthesis of this regulon is induced when Escherichia coli is grown under different extracytoplasmic stress conditions and upon infection by filamentous phage (phage shock) [7, 9, 10, 11, 12, 13, 14].
The activity of PspF is inhibited by PspA, which is an accessory protein that binds directly to PspF to inhibit transcription of the psp regulon [9, 15]. PspA inhibits PspF activity by direct interaction of the AAA+ transcription activation domain of PspF . Although PspA appears to regulate transcription of the psp regulon, it does not bind to DNA , but it is necessary for repressing this regulon .
PspA appears to bind to the W56 loop of PspF, which is a region that contains residues 50-62. This loop contains a group of three amino acids, called the YLW patch, that serve as a docking site for PspA and for ATP hydrolytic activity . The PspF-PspA complex is mainly located in the nucleoid; however, this complex is able to frequently travel from the nucleoid to the membrane to sense changes in the membrane that result from some types of stress .
On the other hand, PspB and PspC together act cooperatively to activate transcription of the psp regulon by blocking the interaction between PspA and PspF [9, 19]. PspC is essential for this activation, whereas PspB is not strictly required . PspA, PspB, and PspC form a complex, and PspC is required for PspA to bind to PspB . PspA, PspB, and PspC are not observed to cross-link with PspD .
The PspF AAA+ domain interacts with σ54 in the presence of a compound that mimics the transition state of ATP hydrolysis . The AAA+ domain may directly contact DNA while promoting open complex formation . Detailed analysis of the role of the C7 and the C3 regions of PspF has been presented with respect to ATP hydrolysis, protein oligomerization, binding to the σ54 holoenzyme, and transcription activation .
A conformational change in PspF is observed in the presence of ATP . ATPase activity may result in transient presentation of the site that interacts with σ54 . Steps leading to open complex formation at the promoter have been examined using PspF and various nucleoside analogs .
Expression of pspF is negatively autoregulated [1, 2, 6, 8, 26]. The stability of pspF mRNA is enhanced by a RIB element (bacterial interspersed mosaic element) in the 3'-flanking region of the pspF transcript .
The PspF protein belongs to the enhancer-binding protein family of σ54-dependent activators (bEBPs). PspF is composed of two domains: the N-terminal catalytic domain, common to members of the AAA+ protein family (ATPase associated with various cellular activities), and a C-terminal helix-turn-helix DNA-binding domain . PspF does not contain a regulatory domain typical for other bEBP proteins; instead, its activity is negatively controlled by PspA acting in trans [15, 16, 27]. The mechano-chemical coupling in bEBPs requires distinct activities of the AAA+ subunits .
The catalytic AAA+ domain is necessary and sufficient to activate σ54 transcription in vivo and in vitro. Hexamerization is required for this function and does not require ATP, DNA, or the DNA-binding domain [3, 29, 30, 31]. When assembled into a ring, the AAA+ domain uses the energy from ATP binding, hydrolysis, and product release to remodel the σ54-RNA polymerase holoenzyme so that it can transition from the closed to the open complex. The AAA+ domain contains all conserved motifs of the AAA+ family, including Walkers A and B for ATP binding and hydrolysis, and the second region of homology (SRH), which contains arginine fingers for intersubunit catalysis [32, 33, 34]. As a member of the bEBP family it carries two additional sequence insertions: the L1 loop containing the GAFTGA motif and the L2 loop (also termed the pre-SIi loop). The L1 loop is known to interact with σ54, presumably with region I of σ54. Zhang et al. (2012) showed that L1 is multifunctional, since it interacts with three elements important for isomerization from RPc to RPo (the RNAP closed and open complexes, respectively). On one hand, L1 contacts the DNA nontemplate strand immediately upstream of the -24 promoter element, and on other hand, it contacts two PspF L1-binding sites, residues 18 to 25 and 33 to 39, within σ54 RI . The GAFTGA motif is thought to communicate changes associated with ATP hydrolysis, leading to conformational rearrangements in the RNA polymerase closed complex and thereby promoting open complex formation [24, 36, 37, 38]. The L2 loop is thought to coordinate movement of the L1 loop [39, 40].
Based on an engineered single-chain polypeptide, it was shown that the PspF hexamer functions asymmetrically, i.e., the individual subunits make different contributions to the activities of the oligomer, and only three subunits are necessary for engagement and remodeling of the target (σ54) of the closed complex (RPc formation) .
Reviews: [11, 12, 42, 43]
|Connectivity class:||Local Regulator|
|Length:||978 bp / 325 aa|
|TU(s) encoding the TF:||
|Regulated gene(s)||pspA, pspB, pspC, pspD, pspE, pspF, pspG|
|Multifun term(s) of regulated gene(s)||
MultiFun Term (List of genes associated to the multifun term)
prophage genes and phage related functions (5)
pspA, pspB, pspC, pspD, pspF
pspA, pspB, pspC, pspF
inhibition / activation of enzymes (1)
cell killing (1)
Transcription related (1)
pspFRead more >
|Regulated operon(s)||pspABCDE, pspF, pspG|
|First gene in the operon(s)||pspA, pspF, pspG|
|Simple and complex regulons|
|Simple and complex regulatory phrases||
Regulatory phrase (List of promoters regulated by the phrase)
|Functional conformation||Function||Promoter||Sigma factor||Central Rel-Pos||Distance to first Gene||Genes||Sequence||LeftPos||RightPos||Evidence (Confirmed, Strong, Weak)||References|
|PspF||activator||pspAp||Sigma54||-119.0||-160.0||pspA, pspB, pspC, pspD, pspE||
|1367911||1367927||[BCE], [BPP], [GEA]||, , |
|PspF||activator||pspAp||Sigma54||-99.0||-140.0||pspA, pspB, pspC, pspD, pspE||
|1367931||1367947||[BCE], [BPP], [GEA]||, , |
|1367931||1367947||[BCE], [BPP], [GEA]||, , |
|1367911||1367927||[BCE], [BPP], [GEA]||, , |
|4262721||4262737||[BPP], [GEA], [ICA]|||
|Evolutionary conservation of regulatory elements|
 Jovanovic G., Rakonjac J., Model P., 1999, In vivo and in vitro activities of the Escherichia coli sigma54 transcription activator, PspF, and its DNA-binding mutant, PspFDeltaHTH., J Mol Biol. 285(2):469-83
 Lloyd LJ., Jones SE., Jovanovic G., Gyaneshwar P., Rolfe MD., Thompson A., Hinton JC., Buck M., 2004, Identification of a new member of the phage shock protein response in Escherichia coli, the phage shock protein G (PspG)., J Biol Chem. 279(53):55707-14
 Jovanovic G., Weiner L., Model P., 1996, Identification, nucleotide sequence, and characterization of PspF, the transcriptional activator of the Escherichia coli stress-induced psp operon., J Bacteriol. 178(7):1936-45
 Amin MA., Awadein MR., Gabr H., 2005, Evaluation of the inhibitory effect of antisense oligodeoxynucleotides on the growth of hepatitis C-associated hepatocellular carcinoma cells in vitro., Chin J Dig Dis. 6(3):142-8
 Dworkin J., Jovanovic G., Model P., 1997, Role of upstream activation sequences and integration host factor in transcriptional activation by the constitutively active prokaryotic enhancer-binding protein PspF., J Mol Biol. 273(2):377-88
 Weiner L., Brissette JL., Model P., 1991, Stress-induced expression of the Escherichia coli phage shock protein operon is dependent on sigma 54 and modulated by positive and negative feedback mechanisms., Genes Dev. 5(10):1912-23
 Jovanovic G., Lloyd LJ., Stumpf MP., Mayhew AJ., Buck M., 2006, Induction and function of the phage shock protein extracytoplasmic stress response in Escherichia coli., J Biol Chem. 281(30):21147-61
 Kobayashi H., Yamamoto M., Aono R., 1998, Appearance of a stress-response protein, phage-shock protein A, in Escherichia coli exposed to hydrophobic organic solvents., Microbiology. 144 ( Pt 2):353-9
 Elderkin S., Jones S., Schumacher J., Studholme D., Buck M., 2002, Mechanism of action of the Escherichia coli phage shock protein PspA in repression of the AAA family transcription factor PspF., J Mol Biol. 320(1):23-37
 Zhang N., Simpson T., Lawton E., Uzdavinys P., Joly N., Burrows P., Buck M., 2013, A key hydrophobic patch identified in an AAA??? protein essential for its in trans inhibitory regulation., J Mol Biol. 425(15):2656-69
 Mehta P., Jovanovic G., Lenn T., Bruckbauer A., Engl C., Ying L., Buck M., 2013, Dynamics and stoichiometry of a regulated enhancer-binding protein in live Escherichia coli cells., Nat Commun. 4:1997
 Chaney M., Grande R., Wigneshweraraj SR., Cannon W., Casaz P., Gallegos MT., Schumacher J., Jones S., Elderkin S., Dago AE., Morett E., Buck M., 2001, Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP-aluminum fluoride: insights into activator mechanochemical action., Genes Dev. 15(17):2282-94
 Cannon WV., Schumacher J., Buck M., 2004, Nucleotide-dependent interactions between a fork junction-RNA polymerase complex and an AAA+ transcriptional activator protein., Nucleic Acids Res. 32(15):4596-608
 Bordes P., Wigneshweraraj SR., Schumacher J., Zhang X., Chaney M., Buck M., 2003, The ATP hydrolyzing transcription activator phage shock protein F of Escherichia coli: identifying a surface that binds sigma 54., Proc Natl Acad Sci U S A. 100(5):2278-83
 Darbari VC., Lawton E., Lu D., Burrows PC., Wiesler S., Joly N., Zhang N., Zhang X., Buck M., 2014, Molecular basis of nucleotide-dependent substrate engagement and remodeling by an AAA+ activator., Nucleic Acids Res. 42(14):9249-61
 Bordes P., Wigneshweraraj SR., Zhang X., Buck M., 2004, Sigma54-dependent transcription activator phage shock protein F of Escherichia coli: a fragmentation approach to identify sequences that contribute to self-association., Biochem J. 378(Pt 3):735-44
 Walker JE., Saraste M., Runswick MJ., Gay NJ., 1982, Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold., EMBO J. 1(8):945-51
 Zhang N., Joly N., Buck M., 2012, A common feature from different subunits of a homomeric AAA+ protein contacts three spatially distinct transcription elements., Nucleic Acids Res. 40(18):9139-52
 Bordes P., Wigneshweraraj SR., Chaney M., Dago AE., Morett E., Buck M., 2004, Communication between Esigma(54) , promoter DNA and the conserved threonine residue in the GAFTGA motif of the PspF sigma-dependent activator during transcription activation., Mol Microbiol. 54(2):489-506
 Zhang N., Joly N., Burrows PC., Jovanovic M., Wigneshweraraj SR., Buck M., 2009, The role of the conserved phenylalanine in the sigma54-interacting GAFTGA motif of bacterial enhancer binding proteins., Nucleic Acids Res. 37(18):5981-92
 Burrows PC., Schumacher J., Amartey S., Ghosh T., Burgis TA., Zhang X., Nixon BT., Buck M., 2009, Functional roles of the pre-sensor I insertion sequence in an AAA+ bacterial enhancer binding protein., Mol Microbiol. 73(4):519-33
 Schumacher J., Joly N., Rappas M., Bradley D., Wigneshweraraj SR., Zhang X., Buck M., 2007, Sensor I threonine of the AAA+ ATPase transcriptional activator PspF is involved in coupling nucleotide triphosphate hydrolysis to the restructuring of sigma 54-RNA polymerase., J Biol Chem. 282(13):9825-33