(190as) Biophysical Model of CsrA-mRNA Interactions Expands Canonical Understanding of the CsrA Global Regulator Protein

Bacterial post-transcriptional regulators provide short timescale control of gene expression, frequently in response to external stresses. For the case of multi-target post-transcriptional regulators, understanding how a regulator recognizes up to hundreds of targets with diverse molecular characteristics can offer insight into predicting and engineering the regulatory network. The CsrA (carbon storage regulator A) post-transcriptional regulator protein can potentially bind upwards of hundreds messenger RNA (mRNA) targets in E. coli and impact their expression by modulating mRNA transcription termination, stability, and/or translation. Importantly, this regulator supports global changes in gene expression linked to stress survival and production of amino acid and fatty acid-derived compounds. Studies have established molecular characteristics that hallmark CsrA-mRNA interactions: (i) consensus binding site sequence, (ii) binding site structural motif, and (iii) preferred number of copies and spacing of binding sites. However, this information has been inferred from a small pool of approximately 15 biochemically well-characterized CsrA-mRNA interactions (evidence of direct interaction in vitro and regulation by reporter assay in vivo). More recently, studies have increased the number of well-characterized targets to over 30 mRNA. Some of these mRNA lack any instance of the established consensus binding site sequence, while others present the sequence without the structural motif or contain an unanticipated number or spacing of consensus binding site sequences.

In order to assess the impact of diverse molecular characteristics of mRNA targets on CsrA interaction and regulation, we developed a thermodynamic, biophysical model of CsrA-mRNA interactions that we term a “canonical” model. It relies on the hallmark characteristics described above to predict the (i) identity, (ii) binding free energy, and (iii) effect of CsrA binding on translation (via the RBS Calculator) of an ensemble of potential CsrA binding sites within each mRNA. After establishing that binding site and binding free energy predictions are supported by literature footprints and fluorescent reporter assays, respectively, we apply the canonical model to a pool of 107 mRNA: 32 are well-characterized targets, while the remainder show regulation by CsrA in fluorescent reporter assays but direct interaction has not yet been confirmed. The model captures regulation of 62% of tested mRNA targets, implying that unaccounted for mechanisms may govern regulation of remaining 41 mRNA. Close analysis of ensembles of predicted binding sites reveals that CsrA may bind elongated nucleotide stretches rich in consensus-like sequences in addition to specific copies of the consensus sequence to regulate mRNA expression. This expanded understanding of CsrA-mRNA recognition may begin to account for the diversity of molecular characteristics observed among its mRNA targets and its wide scope of cellular effects.

This abstract details work contained in a manuscript currently under review: Leistra, A. N., Gelderman, G., Sowa, S. W., Moon-walker, A., Salis, H. M., and Contreras, L. M. A Canonical Biophysical Model of the CsrA Global Regulator Suggests Flexible Regulator-Target Interactions. 2018.