Structure and Mechanism of SAM-responsive Riboswitches

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DESCRIPTION (provided by applicant): Non-coding RNA is known to play crucial roles at almost every level of the maintenance and transmission of biological information. These RNAs and their assemblies into ribonucleoproteins (RNPs) perform diverse tasks such as maintaining the ends of chromosomes, X-chromosome inactivation, processing and modification of pre-RNAs, and the targeting of proteins to specific cellular locations. My research program focuses on understanding the relationship between non-coding RNA structure and function. In this proposal, we aim to study a class of non-coding RNAs called riboswitches, cis-acting elements found in the 5'-untranslated region (5'-UTR) of bacterial mRNAs that regulate gene expression via their ability to directly bind small molecule metabolites. These riboregulatory elements control a variety of basic metabolic pathways in a number of pathogenic bacteria, including B. anthracis, S. aureus and M. tuberculosis; in Bacillus species, over 2% of all genes are controlled in this fashion. Sulfur metabolism is one of the most important aspects of cellular metabolism controlled by riboswitches, which is effected through direct interaction of S-adenosylmethionine (SAM) with four distinct subclasses of SAM-responsive RNAs. To develop a detailed structural and biochemical understanding of these SAM-responsive riboswitches, we have solved the structure of two separate subclasses using X-ray crystallography. Building from this work, we propose to use a combination of X-ray crystallography, binding studies and chemical probing to address: (1) what is the structural basis for SAM recognition, (2) how does RNA effectively discriminate between SAM and the product form S- adenosylhomocysteine (SAH), (3) what are the conformational changes in the RNA that accompany ligand binding, and (4) how are these conformational changes used to effect gene regulation. The results of these proposed studies will serve to broaden our knowledge of RNA-based gene regulation as well as provide an atomic-level understanding of an RNA that is a promising antimicrobial therapeutic target. PUBLIC HEALTH RELEVANCE: Riboswitches are a form of RNA-based gene regulation that is widely utilized in bacteria, including a number of medically important pathogenic bacteria such as B. anthracis, M. tuberculosis, P. aeruginosa and S. aureus. Our work seeks to develop an atomic-level understanding of how these RNAs regulate sulfur metabolism through their ability to directly bind S-adenosylmethionine. These studies serve to develop these RNAs as potential targets of antibacterial therapeutics via structure-based drug design.