Abstract
BackgroundTo survive, bacteria must be able to adapt to environmental stresses. Small regulatory RNAs have been implicated as intermediates in a variety of stress-response pathways allowing dynamic gene regulation. The RNA binding protein Hfq facilitates this process in many cases, helping sRNAs base pair with their target mRNAs and initiate gene regulation. Although Hfq has been identified as a critical component in many RNPs, the manner by which Hfq controls these interactions is not known.Methodology/Principal FindingsTo test the requirement of Hfq in these mRNA-sRNA complexes, the OxyS-fhlA system was used as a model. OxyS is induced in response to oxidative stress and down regulates the translation of fhlA, a gene encoding a transcriptional activator for formate metabolism. Biophysical characterization of this system previously used a minimal construct of the fhlA mRNA which inadvertently removed a critical element within the leader sequence of this mRNA that effected thermodynamics and kinetics for the interaction with Hfq.Conclusions/SignificanceHerein, we report thermodynamic, kinetic and structural mapping studies during binary and ternary complex formation between Hfq, OxyS and fhlA mRNA. Hfq binds fhlA mRNA using both the proximal and distal surfaces and stimulates association kinetics between the sRNA and mRNA but remains bound to fhlA forming a ternary complex. The upstream Hfq binding element within fhlA is similar to (ARN)x elements recently identified in other mRNAs regulated by Hfq. This work leads to a kinetic model for the dynamics of these complexes and the regulation of gene expression by bacterial sRNAs.
Highlights
Small non-coding RNAs mediate gene regulation in both bacteria and eukaryotes [1,2,3]
OxyS and Hfq interacts with fhlA leader construct to form a ternary complex To test whether the upstream leader region of fhlA facilitates the interaction with OxyS and Hfq as previously described for the rpoS-DsrA system [23], the leader sequence of fhlA was extended from the previously characterized position 253 relative to the start codon, to 2136 and 2220 respectively
In addition to its function of promoting base pairing of Small non-coding RNAs (sRNA) to their target mRNAs, Hfq is thought to engage ribosomes, poly A polymerase and RNase E and other enzymes that are involved in RNA transactions [11]
Summary
Small non-coding RNAs (sRNA) mediate gene regulation in both bacteria and eukaryotes [1,2,3]. Cis RNAs derive from the same genetic locus as the regulated message but are transcribed from the antisense strand; exhibiting perfect complementarity with their target. These RNAs are known to control regulatory pathways such as transcriptional attenuation, RNA processing and decay, and translation initiation [5,6]. Unlike cis-acting sRNAs, the trans-acting sRNAs are expressed from genetic loci different than their targets and interact using imperfect base pairing. These sRNAs often require accessory proteins such as Hfq for activity. Hfq has been identified as a critical component in many RNPs, the manner by which Hfq controls these interactions is not known
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