StpA and Hha stimulate pausing by RNA polymerase by promoting DNA–DNA bridging of H-NS filaments

Abstract

In enterobacteria, AT-rich horizontally acquired genes, including virulence genes, are silenced through the actions of at least three nucleoid-associated proteins (NAPs): H-NS, StpA and Hha. These proteins form gene-silencing nucleoprotein filaments through direct DNA binding by H-NS and StpA homodimers or heterodimers. Both linear and bridged filaments, in which NAPs bind one or two DNA segments, respectively, have been observed. Hha can interact with H-NS or StpA filaments, but itself lacks a DNA-binding domain. Filaments composed of H-NS alone can inhibit transcription initiation and, in the bridged conformation, slow elongating RNA polymerase (RNAP) by promoting backtracking at pause sites. How the other NAPs modulate these effects of H-NS is unknown, despite evidence that they help regulate subsets of silenced genes in vivo (e.g. in pathogenicity islands). Here we report that Hha and StpA greatly enhance H-NS-stimulated pausing by RNAP at 20°C. StpA:H-NS or StpA-only filaments also stimulate pausing at 37°C, a temperature at which Hha:H-NS or H-NS-only filaments have much less effect. In addition, we report that both Hha and StpA greatly stimulate DNA–DNA bridging by H-NS filaments. Together, these observations indicate that Hha and StpA can affect H-NS-mediated gene regulation by stimulating bridging of H-NS/DNA filaments.

Structural basis for osmotic regulation of the DNA binding properties of H-NS proteins

“…In contrast, StpA, an H-NS homologue, requires no or sub mM amounts of MgCl 2 to be able to bridge DNA 52, 53 . The high sensitivity of StpA towards magnesium chloride is possibly due to the weaker electrostatic interdomain interactions compared to H-NS and MvaT, as concluded from the charge distribution within its primary sequence (fig.…”

Post-translational modification of nucleoid-associated proteins: an extra layer of functional modulation in bacteria?

“…Interestingly, this lysine is subject to acetylation [64], which could reduce the DNA-binding affinity of StpA to resemble that of H-NS. In turn, such a modification could reduce the extent to which StpA enhances bridging through the formation of H-NS-StpA bridging filaments [30]. StpA outperforms H-NS in binding RNA as well, both being involved in posttranscriptional regulation of malT and td expression [68,69].…”

“…If interaction between H-NS and StpA involves a comparable salt bridge, such a PTM may decrease the possibility of an H-NS-StpA interaction. Modulation of oligomerization along DNA and DNA bridging are key aspects of H-NS-mediated modulation of gene expression [24,30]. Thus, these PTMs could allow bacteria to regulate transcription, thereby adapting to their environment.…”

“…Owing to dimer-dimer interactions, the protein forms filaments along DNA [26]; its multivalency also facilitates bridging of two DNA duplexes [27,28], potentially yielding loops in vivo [29]. The DNA-binding properties of H-NS and its various roles are modulated by interaction with a paralogue, StpA, association partners (such as Hha and YdgT) and changes in physicochemical conditions (such as osmolarity) [24,[30][31][32].…”

Growth Phase Dependent Chromosome Condensation and H-NS Protein Redistribution in E. coli Under Osmotic Stress

“…Notably, StpA demonstrates higher affinity for DNA in vitro in the presence of salt 29,30 . To assess whether StpA plays any role in displacing H-NS from the nucleoid we visualized H-NS-mEos localization in an isogenic strain harbouring a deletion in stpA.…”