YloC, a new ribonuclease of Bacillus subtilis

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.

Grant Number: 5R01GM147211-03
NIH Institute/Center: NIH
Principal Investigator: DAVID BECHHOFER