The F plasmid transfer region is regulated by an intricate web of host- and plasmid-encoded factors, with F TraJ and H-NS playing important opposing roles in regulating F transfer region gene expression in response to nutritional and extracytoplasmic stress (Will et al., 2004; Lau-Wong et al., 2008; Frost & Koraimann, 2010). However, the mechanism by which F TraJ counteracts H-NS repression remains unclear. F TraJ appears to contain an HTH DNA-binding motif (residues 154–180), suggesting that TraJ and H-NS might compete for DNA-binding sites within the PY region. F TraJ

contains a glycine (G166) at the turn between helix-2 and helix-3, the recognition helix, which is characteristic of HTH DNA-binding proteins (Pabo & Sauer, 1992; Aravind et al., 2005). Mutations this website of G166, Y163 and H169 within the HTH motif resulted in reduced mating ability using complementation assays, whereas mutations upstream or PD0325901 downstream of the motif did not affect mating ability. This would suggest that, whereas the glycine is important, the sequence of the helices within the HTH motif can vary. The importance of G166 for DNA binding was revealed using the ChIP assay. Although this assay did not indicate the precise

sequence recognized by TraJ, it demonstrated that TraJ is a DNA-binding protein and that it binds to the PY region and potentially releases it from H-NS silencing (Will & Frost, 2006). The deletion of only four amino acids from the C-terminus 17-DMAG (Alvespimycin) HCl of TraJ prevented the activation of PY as measured by mating ability assays, but did not prevent TraJ dimerization or DNA binding in vivo. Thus, desilencing of H-NS-repressed PY by F TraJ appears to involve other aspects of TraJ function. Remarkably, deletion of the last four amino acids from the Yersinia pseudotuberculosis activator RovA, which counteracts H-NS silencing of the inv genes in that system, also blocks RovA function, but does not prevent its binding to DNA (Tran et al., 2005). TraJ, RovA and a similar activator in Salmonella enterica, SlyA (Perez et al., 2008), share sequence similarity and charge distribution within their

C-terminal tails (Fig. 1b). Nevertheless, it seems that charge is not an important factor in TraJ or RovA functioning because single substitutions of charged C-terminal amino acids by alanine did not have any effect on transcriptional activation. The C-terminal tail in RovA is considered to be surface exposed in order to interact with RNA polymerase and directly activate transcription (Tran et al., 2005). RovA and SlyA are members of the MarR/SlyA subfamily that are homodimers (Ellison & Miller, 2006) and bind DNA via a winged-helix domain, which is an HTH motif, followed by two β-strands (Aravind et al., 2005; Fang & Rimsky, 2008). Although it more closely resembles tetra-helical HTH proteins such as LuxR (Aravind et al., 2005), TraJ might activate transfer gene expression in a manner similar to RovA and SlyA.