Poster abstracts

Poster number 8 submitted by Ian Hall

Investigating Natural Variation in SAM-I Riboswitch Structure and Function

Ian Hall (Department of Chemistry, University of Michigan), Kate Zablock (Program in Biophysics, University of Michigan), Raeleen Sobetski (Program in Biophysics, University of Michigan), Sarah Keane (Department of Chemistry and Program in Biophysiscs, University of Michigan)

Methionine and its metabolites are essential for the growth and development of all organisms. Mechanisms for the recognition and regulation of methionine levels are diverse among the domains of life. In many bacteria, the metabolite S-adenosyl methionine (SAM) is recognized by SAM-I riboswitches which control the transcription of methionine biosynthesis and transport genes in a SAM-dependent manner. SAM-I riboswitches are non-coding (nc)RNAs found in the 5 ′ untranslated region (UTR) of some bacterial messenger (m)RNAs. SAM-I riboswitches bind SAM within an aptamer domain which directs the co-transcriptional folding of an expression platform. When SAM is bound, a terminator hairpin forms, causing premature termination of transcription. Without SAM, the expression platform folds into an anti-terminator hairpin that permits transcription of downstream genes.

Previous work in Bacillus subtilis discovered natural variation in the ability of SAM-I riboswitches to bind SAM and terminate transcription.1 Seven candidate SAM-I riboswitches were identified in Listeria monocytogenes and named SAM riboswitch elements A-G (SreA-G).2 These candidate riboswitches have yet to be functionally validated but are predicted to fold into the conserved SAM-I riboswitch secondary structure. The current work is a robust biochemical and biophysical characterization of the candidate riboswitches. The ligand affinity and specificity of the riboswitches were determined by isothermal titration calorimetry (ITC). The ability and efficiency of the riboswitches to terminate transcription were evaluated by a radiolabeled transcription termination assay. The secondary and tertiary structures of the riboswitches were investigated by chemical probing and small-angle X-ray scattering (SAXS), respectively. The goal of this work is to functionally validate SreA-G as SAM-I riboswitches and to understand how natural variance in the riboswitches' ability to bind SAM and terminate transcription is related to riboswitch structure and genomic location.

1. Tomšič et al. Natural Variability in S-Adenosylmethionine (SAM)-Dependent Riboswitches: S-Box Elements in Bacillus subtilis exhibit Differential Sensitivity to SAM In Vivo and In Vitro. Journal of Bacteriology. 2008.
2. Toledo-Arana et al. The transcriptional landscape of Listeria monocytogenes: switch from saprophytism to virulence. Nature. 2009.

Keywords: Riboswitch, RNA Structure, Molecular Recognition