Abstract
Bacterial global post-transcriptional regulators execute hundreds of interactions with targets that display varying molecular features while retaining specificity. Herein, we develop, validate, and apply a biophysical, statistical thermodynamic model of canonical target mRNA interactions with the CsrA global post-transcriptional regulator to understand the molecular features that contribute to target regulation. Altogether, we model interactions of CsrA with a pool of 236 mRNA: 107 are experimentally regulated by CsrA and 129 are suspected interaction partners. Guided by current understanding of CsrA-mRNA interactions, we incorporate (i) mRNA nucleotide sequence, (ii) cooperativity of CsrA-mRNA binding, and (iii) minimization of mRNA structural changes to identify an ensemble of likely binding sites and their free energies. The regulatory impact of bound CsrA on mRNA translation is determined with the RBS calculator. Predicted regulation of 66 experimentally regulated mRNAs adheres to the principles of canonical CsrA-mRNA interactions; the remainder implies that other, diverse mechanisms may underlie CsrA-mRNA interaction and regulation. Importantly, results suggest that this global regulator may bind targets in multiple conformations, via flexible stretches of overlapping predicted binding sites. This novel observation expands the notion that CsrA always binds to its targets at specific consensus sequences.
Highlights
For the case of CsrA, approximately 800 mRNA have been identified across multiple environmental conditions as potentially interacting with CsrA2,5: this total approaches 20% of the E. coli genome
We developed a biophysical model of canonical CsrA binding and regulation to investigate how the molecular features of a target mRNA can influence its regulation (Figs 1 and 2)
After establishing that the model correctly predicted the regulatory modes of 66 of 107 mRNA targets with known CsrA regulation (Table 2), we demonstrated the ability of the model to (i) correctly predict CsrA binding site locations across a range of binding affinities (Figs 4 and 5)
Summary
For the case of CsrA, approximately 800 mRNA have been identified across multiple environmental conditions as potentially interacting with CsrA2,5: this total approaches 20% of the E. coli genome. The CsrA homodimer binds a target mRNA at two copies[17] of a consensus sequence (ANGGA)[18], preferentially located in the loop of a hairpin structure[18,19]. Among the 31 classical and well-characterized targets of CsrA (defined in Table 1), mRNAs like pgaA may hold to the general pattern[20], but clpB, dps, patA, and purM do not present the consensus ANGGA binding motif[5]. Hfq and ycdT present only a single copy of the consensus sequence[20,21,22,23,24] and cstA21 presents footprinted binding sites outside of the typical stem loop structures that have been shown favorable to CsrA binding
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