Control of bacterial gene expression by the FinO family of RNA chaperones

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Background: Small non-coding RNAs (sRNAs) regulate gene expression in essentially all bacterial species with the assistance of proteins called RNA chaperones. The FinO family of RNA chaperones is one of the most important and widespread chaperone families with roles in regulation of bacterial stress responses and horizontal gene transfer (HGT). Our collaborator, Xavier Charpentier (Lyon) discovered a new FinO chaperone in Legionella called RocC. His group showed that RocC binds a single sRNA called RocR, thereby stabilizing RocR and facilitating the repression of specific mRNA targets that control HGT. We set out to understand how RocC specifically binds RNA and how it facilitates sRNA-mRNA pairing. We showed that the RocC FinO domain binds the hairpin-tail transcription terminator of RocR and we went on to determine the crystal structure of the RocC FinO domain in complex with a RocR terminator. This structure provides the first view of any FinO chaperone bound to its RNA and provides the basis for our key hypotheses that will guide our experiments in the next term of this program. Hypothesis: We propose that FinO chaperones bind transcriptional terminator structures in a manner that reads the length of the 3' ssRNA tail. In addition, we propose that the mechanism of binding allows the remodeling of the sRNA to enable sRNA-mRNA pairing. Specific Aim 1. Refining our understanding of RocC-RocR interactions. To understand the functional implications of our RocC-RocR structure, we are constructing a large set of site-directed point mutations in RocC as well as modifications in the RocR hairpin-tail to understand critical interactions for binding affinity/specificity. The effects of these mutations will be probed through biochemical and biophysical assays, and their effects on RocR stabilization, gene expression and competence will be assessed in Legionella. Specific Aim 2. Probing the effects of RocC binding on RocR structure and interactions with mRNA targets. In preliminary work, we showed that RocR adopts a pseudoknot structure between the terminator hairpin and the sRNA "seed" sequence, and we propose that this must unfold to facilitate sRNA-mRNA pairing. We will test this idea through footprinting, NMR studies, crystallization of the pseudoknot structure and analysis of larger scale RocC-RocR structures via small angle X-ray scattering. These hypotheses will be further tested through in vivo studies in Legionella. Specific Aim 3. Understanding the breadth of FinO chaperone function in bacteria. Using a set of FinO chaperones that span the FinO phylogenetic tree, we will compare their structures, RNA binding specificities, and RNA pairing activities to understand the degree of conservation of FinO functions, and these roles will be further tested in domain-swap experiments. These experiments should reveal the basis for specificity differences between these chaperones, as well as how these proteins facilitate RNA-RNA pairing.