Defining the roles of small KH-domain RNA binding proteins in mycobacteria

Project summary Mycobacteria include important human pathogens such as Mycobacterium tuberculosis, and elucidating the mechanisms by which they regulate gene expression is necessary to understand their mechanisms of surviving drugs and other stressors encountered during infection. However, the mycobacteria are evolutionarily divergent from better-studied model organisms. Small RNA binding proteins have critical roles in mediating sRNA-mRNA interactions and modulating mRNA stability in E. coli. However, mycobacteria lack orthologs of the RNA binding proteins that are best characterized in E. coli. Instead, mycobacteria encode two putative RNA binding proteins, KhpA and KhpB, that have orthologs in some gram-positive bacteria where their functions are poorly understood. Given the almost complete absence of published data on small RNA binding proteins in mycobacteria, we propose to determine the biochemical properties and functional impact of KhpA and KhpB using the non-pathogenic model mycobacterial species Mycolicibacterium smegmatis. The proposed project will address a major knowledge gap in the mycobacterial field while providing in-depth training opportunities for undergraduate students at Worcester Polytechnic Institute. We hypothesize that KhpA and KhpB form both homodimers and heterodimers that bind differing suites of mRNAs and small regulatory RNAs (sRNAs), affecting their stability and function. The objectives of this proposal are to determine the functional oligomerization states of these two proteins; determine the compendia of RNAs that they bind; identify the properties that make these RNAs targets of KhpA/B; determine the impact of khpA/B on RNA degradation rates; and determine the impact of khpA/B on growth and stress tolerance.