The role of the conserved phenylalanine in the σ54-interacting GAFTGA motif of bacterial enhancer binding proteins

Abstract

σ54-dependent transcription requires activation by bacterial enhancer binding proteins (bEBPs). bEBPs are members of the AAA+ (ATPases associated with various cellular activities) protein family and typically form hexameric structures that are crucial for their ATPase activity. The precise mechanism by which the energy derived from ATP hydrolysis is coupled to biological output has several unknowns. Here we use Escherichia coli PspF, a model bEBP involved in the transcription of stress response genes (psp operon), to study determinants of its contact features with the closed promoter complex. We demonstrate that substitution of a highly conserved phenylalanine (F85) residue within the L1 loop GAFTGA motif affects (i) the ATP hydrolysis rate of PspF, demonstrating the link between L1 and the nucleotide binding pocket; (ii) the internal organization of the hexameric ring; and (iii) σ54 interactions. Importantly, we provide evidence for a close relationship between F85 and the −12 DNA fork junction structure, which may contribute to key interactions during the energy coupling step and the subsequent remodelling of the Eσ54 closed complex. The functionality of F85 is distinct from that of other GAFTGA residues, especially T86 where in contrast to F85 a clean uncoupling phenotype is observed.

Regulation of Type VI Secretion Gene Clusters by σ 54 and Cognate Enhancer Binding Proteins

“…Interactions of a bEBP and a 54 from two distinct microorganisms have already been demonstrated (50). This is made possible by the high degree of conservation between the 54 binding region of E. coli bEBPs and the bEBPs used in this study: the central domain, including the GAFTGA sequence, which mediates bEBP binding to 54 , is conserved among all the bEBPs used in this study (8,43,49,65) (see Fig. S2 in the supplemental material).…”

Nucleotide-induced asymmetry within ATPase activator ring drives σ54–RNAP interaction and ATP hydrolysis

“…Extensive mutagenesis studies suggest that bEBPs remodel s54 protein via interactions with region I, the protein's N-terminal domain (Gonzalez et al 1998;Bordes et al 2004;Dago et al 2007;Schumacher et al 2007;Zhang et al 2009). Region I spans ;50 amino acids that are predicted to form an a helix; it possesses two short motifs that are suggested to make direct contact with the GAFTGA motifs of the ATPase (Merrick 1993;Zhang et al 2012).…”

The bacterial enhancer-dependent RNA polymerase

“…The sigma54 can be divided into three regions based on sequence and function, although they have varying degrees of functional similarities to the regions of sigma70 and no sequence similarity (Figure 1A,B). In sigma54, Region I (RI, residues 1–56, E. coli numbering) helps maintain the closed promoter complex by inhibiting the DNA melting reaction, includes the major binding site for the bEBPs, and directs the formation of a DNA fork junction structure at the base pair immediately downstream of the promoter −12 element [19,20,30]. Notably, deletion of RI can bypass the need for the ATPase-driven remodelling, provided that the promoter DNA is melted out [11,31–33], which is correlated with loss of binding tightly to the −12 fork junction structure.…”

“…It is notable that the sigma54 polypeptide chain snakes back and forth through its loop regions embedded in the RNAP, and so is not co-linear with the RNA polymerase in a promoter 5′ to 3′ sense (Figure 1C). Consequently, RI interacts with RIII ELH/HTH forming a distinct structural module (Figure 1B) that would correspond to the −12 proximal regulatory centre inferred from biochemical work [20,35–38]. The presence of this regulatory centre also reflects the finding that mutations in RI and RIII can bypass the usual bEBP requirements for making open promoter complexes, presumably through impacts on the networks of interactions made within sigma54, −12 promoter DNA, and their interfaces with the core RNA polymerase.…”

“…A range of genetic and biochemical studies have established some special properties of the sigma54 containing RNA polymerase, which distinguish it from the sigma70-type holoenzyme, and which can be attributed directly to the sigma factor itself. These include the ability of the sigma54 factor in the absence of the core RNA polymerase to bind promoter DNA and to be remodelled by its cognate bEBPs in an ATPase reaction, and equivalent activities are not evident with the sigma70 factor [6–20]. The lack of any significant sequence similarity between sigma54 and sigma70 was noted early on and is borne out by their very different molecular structures and differing modes of action within the core RNA polymerase as described below.…”