PROJECT SUMMARY: Our laboratory has, for many years, studied the essential process of mRNA decay in the model Gram-positive bacterium, Bacillus subtilis. We have identified several ribonuclease (RNase) enzymes of B. subtilis and have elucidated the role they play in mRNA turnover. The viability of a B. subtilis strain lacking all of the known 3’-to-5’ exoribonucleases prompted us to pursue identification of additional RNase activities. Using classic protein biochemistry, we recently identified a novel RNase, named YloC. YloC is an endoribonuclease with a hexameric structure, an unusual characteristic that is shared with only one other RNase: the Nsp15 protein of the SARS-CoV family. Initial experiments suggest that, although YloC has ribonuclease activity in vitro, it may function as an adapter for RNA interactions in vivo. Although proteins with significant homology to YloC are widespread in bacterial species, there is no published information on the structure of any member of this protein family. The current proposal seeks to elucidate the structure and function of YloC, as follows: • Mutagenize highly conserved residues of YloC to determine the effect on several properties – including ribonuclease activity, RNA binding, and structure – and to clarify functional domains of the protein. • Identify high-affinity RNA ligands of YloC via SELEX procedures with random-sequence oligonucleotides and with genomic RNA sequences. • Characterize how the strong interaction of YloC with E. coli polynucleotide phosphorylase (PNPase) acts in small RNA (sRNA) regulation in E. coli and possibly in B. subtilis. • Determine the three-dimensional structure of the highly homologous E. coli YicC protein bound to an RNA substrate, as well as the structure of YloC and/or its homologs from thermophilic bacterial species. This work will build on an initial determination of the structure of YicC. RELEVANCE: Ribonucleases play essential roles in RNA turnover and processing. A thorough understanding of the proteins that bind to and act enzymatically on RNA molecules will enable design of antimicrobial agents that disrupt such proteins and thereby interfere with bacterial cell growth.

PROJECT NARRATIVE: Ribonucleases are enzymes that act on RNAs to process them to their mature, functional form, or to degrade them when they are no longer needed by the cell. A newly discovered ribonuclease in the model microorganism, Bacillus subtilis, is the prototype for a large family of similar proteins that are found in many bacterial organisms. Elucidating the structure and function of this ribonuclease could lead to the design of antibiotics that inhibit this enzyme and thereby reduce bacterial virulence.