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Fall 2011


The Crystallization of Riboswitches: Potential Targets for Antibiotics Artem Serganov, ‘12

Supervisor: Dr. Dinshaw J. Patel, Ph.D., Department of Structrual Biology Memorial Sloan-Kettering Cancer Center 1275 York Avenue, New York, NY 10065

Introduction Widespread antibiotic resistance in bacteria is rapidly developing, making many modern antibiotics ineffective in interfering with the functions of proteins essential for cellular processes in bacteria. This necessitates the development of novel classes of antibiotics specific for new molecular targets. The recent discoveries of RNA interference and other RNA-based regulation systems highlight the importance of RNA in controlling gene expression. Riboswitches are the latest addition to the growing field of RNA-based control elements [1,2] with the unique ability to sense concentrations of metabolites without protein assistance and to direct expression of genes involved in the synthesis and transport of these molecules. Riboswitches are RNA sequences typically located in the 5´ untranslated regions (UTR) of messenger RNAs (mRNA) [3]. Riboswitches usually consist of sensing domains, which specifically bind to metabolites, and expression platforms, which carry gene expression signals. Riboswitches exist in two alternative conformations: Bound or not bound to a metabolite, determining whether gene expression is turned on or off [4]. In the metabolite-bound form the ribosome binding site (RBS) of some riboswitches

becomes inaccessible to ribosomes and gene expression is turned off (Figure 1a). When the riboswitch is in a non-bound state, the ribosome can bind to the RBS and proceed with translation. Depending on the expression platform, riboswitches can control gene expression by translational (via RBS) or transcriptional (via transcription termination) (Figure 1b) mechanisms. Riboswitches can interact with different types of small molecules including co-enzymes, amino acids, nucleobases, magnesium cations, and sugar derivatives [5,6]. Since riboswitches control expression of genes essential for viability of many pathogenic bacteria, and since they have not yet been found in humans, they represent potentially good targets for new antibiotics5. Moreover, riboswitches are naturally adept at binding small druglike molecules that can be used as the foundation for the design of new antibiotics. Bacteria and humans share many metabolites, therefore, to avoid toxicity in humans, metabolite-like drugs have to be different from the natural version but nevertheless capable of binding to the riboswitches and shutting down gene expression. Interestingly, several metabolite-like compounds such as pyrithiamine7 and L-aminoethylcysteine [8,9], utilized for years to combat microbes, likely target

riboswitches. Although new metabolite-like drugs can be found by screening large combinatorial libraries, this process can be facilitated by knowing three-dimensional (3-D) structures of riboswitches bound to their ligands. Many laboratories have already begun efforts to determine the 3-D structures of various riboswitch-ligand complexes, primarily through X-ray crystallography. However the progress of these studies is slow [6,10]. One of the major obstacles in X-ray crystallography is crystallization, which first requires extensive trial-and-error screenings of crystallization conditions, and then necessitates very careful optimization of initial conditions. Therefore, the goal of my project was to produce crystals of two riboswitches specific to S-adenosylmethionine (SAM) and pre-queuosine1 (preQ1) for the determination of their 3-D structures. The SAM-II riboswitch specifically recognizes SAM and negatively controls expression of the bacterial metA gene, which encodes the protein that catalyzes the first step of methionine synthesis [11] (Figure 2a,b). Interestingly, three riboswitches (SAM-I, SAM-II and SAM-III) with different sequences and secondary structures recognize SAM1115, potentially featuring similar SAM binding pockets. If a SAM-like drug is

Figure 1. Example of riboswitch-mediated control of gene expression. In both cases, translation repression (a) and transcription termination (b), the binding of a metabolite (red M) stabilizes the riboswitch structure.

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Fall 2011  

Fall 2011