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, 16]. PspA inhibits PspF activity by direct interaction of the AAA+ transcription activation domain of PspF [15]. Although PspA appears to regulate transcription of the psp regulon, it does not bind to DNA , but it is necessary for repressing this regulon [17]. 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 [18]. 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 [19]. 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, 20]. PspC is essential for this activation, whereas PspB is not strictly required [9]. PspA, PspB, and PspC form a complex, and PspC is required for PspA to bind to PspB [21]. PspA, PspB, and PspC are not observed to cross-link with PspD [21]. The PspF AAA+ domain interacts with σ⁵⁴ in the presence of a compound that mimics the transition state of ATP hydrolysis [22]. The AAA+ domain may directly contact DNA while promoting open complex formation [23]. 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 [24]. A conformational change in PspF is observed in the presence of ATP [24]. ATPase activity may result in transient presentation of the site that interacts with σ⁵⁴ [25]. Steps leading to open complex formation at the promoter have been examined using PspF and various nucleoside analogs [26]. Expression of pspF is negatively autoregulated [2, 4, 7, 8, 27]. The stability of pspF mRNA is enhanced by a RIB element (bacterial interspersed mosaic element) in the 3'-flanking region of the pspF transcript [27]. 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 [7]. PspF does not contain a regulatory domain typical for other bEBP proteins; instead, its activity is negatively controlled by PspA acting in trans [15, 17, 28]. The mechano-chemical coupling in bEBPs requires distinct activities of the AAA+ subunits [29]. 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, 30, 31, 32]. 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 [33, 34, 35]. 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 RP_c to RP_o (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 [36]. 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 [25, 37, 38, 39]. The L2 loop is thought to coordinate movement of the L1 loop [40, 41]. 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 (RP_c formation) [42]. Reviews: [11, 12, 43, 44]