Delineating the mechanism of RelA mediated Hfq multimerization

The highly conserved RNA binding protein Hfq has been known to be an important mediator of post-transcriptional regulation of gene expression. It is a chaperone that helps in sRNA-mRNA base pairing. During iron depletion, the small RNA RyhB functions to regulate the expressions of various iron storage and iron containing proteins. As many base pairing RNAs, this regulation requires the function of Hfq.

Previously, we showed that apart from Hfq, regulation of a number of target genes of RyhB required the function of RelA, the highly conserved stringent response regulator. We showed that RelA enhanced Hfq oligomerization, which in turn resulted in efficient binding to RyhB (Argaman et al., 2012). RelA harbors 2 domains: a N- terminal synthetase domain and a C- terminal regulatory domain. To further characterize which of the domains of RelA is responsible for Hfq multimerization we carried out deletion mapping and random mutagenesis in the relA gene. In vivo and in vitro studies of the deletion and the single point mutants indicated that specific sites in the N terminal domain of RelA are active in Hfq multimerization. We also performed point mutations in proximal and distal regions of the hfq gene and found that initial RNA binding to Hfq is essential for RelA to mediate its action on Hfq, which also supports our current hypothesis involving RNA as the mediator in between RelA and Hfq. Based on our studies we propose that RelA triggers the assembly of RNA bound Hfq dimers to form higher oligomeric forms.