The goal of this project is to define the function of biologically important noncoding RNAs in Mycobacterium tuberculosis. It has recently become clear that noncoding RNAs play important regulatory roles in many prokaryotes. However, in Mtb, like most bacteria, the vast majority of them remain completely uncharacterized. We have experimentally identified over 1000 noncoding RNAs in Mtb, of which only 5 have been experimentally characterized to any extent. In this proposal, we will focus on one class of noncoding RNAs, trans-acting, intergenic regulatory RNAs, commonly termed small RNAs (sRNAs). While some sRNAs interact with and affect the function of proteins, the vast majority of sRNAs characterized to date influence the stability and/or translation of mRNAs. Thus, the critical steps in defining the biological function a regRNA are to identify the mRNA(s) it regulates and the biological consequences of this regulation—first at the level of the transcriptome and proteome (regulon) and then in terms of the cell's physiology. We propose a tiered series of aims in which we will define the function of candidate sRNAs with increasing specificity—first defining the importance of the candidates for Mtb growth under standard and "in vivo-like" growth conditions. We will then define the regulatory network controlled by each sRNA. Finally, we will use a combination of computational and biochemical approaches to identify and validate the target of each sRNA. We anticipate that through these studies, we will provide unprecedented insights into sRNA function in bacteria in general and in Mtb more specifically.

RELEVANCE (See instructions): Mycobacterium tuberculosis remains a catastrophic global health problem. In this proposal, we seek to define the function of a novel class of genes, regulatory RNAs, which have only recently been discovered in prokaryotes. We anticipate that the discoveries from these fundamental studies may ultimately lead to a better understanding of how to develop effective drugs and vaccines for tuberculosis.