Poster abstracts
Poster number 142 submitted by Jiarui Wang
Probing the effect of different ligands on the conformational dynamics of a transcriptionally acting preQ1 riboswitch using single molecule FRET microscopy
Jiarui Wang (Department of Chemistry, University of Michigan), Krishna Chaitanya Suddala (Department of Biophysics, University of Michigan)
Abstract:
Non-coding RNAs play crucial roles in a multitude of biological processes such as translation, messenger RNA (mRNA) splicing and regulation of gene expression. Riboswitches are regulatory elements found mainly in the 5’ untranslated regions of numerous bacterial mRNAs capable of modulating gene expression in response to the binding of cellular metabolites. Riboswitches
comprise two functional components: a ligand binding aptamer domain and an expression platform whose conformation decides the fate of gene expression. Despite its smallest size among all reported aptamers, the Bacillus subtilis (Bsu) preQ1 (pre-queuosine) riboswitch boasts precise control of the expression of genes involved in the biosynthesis of queuosine. The co-crystal structure of the Bsu aptamer domain bound to its ligand preQ1 showed a pseudoknot structure. However, the nature of folding pathway remained elusive, until recently. We have recently used single molecule fluorescence resonance energy transfer (smFRET) and computational simulations to show that the ligand-free Bsu aptamer adopts a pre-folded conformation in which the single stranded A-rich tail interacts with stem-loop P1-L1. Previous studies indicated that the Bsu riboswitch binds closely related ligands preQ0 and guanine with similarly favorable affinities. The effects of these ligands on the conformational dynamics, however, were not previously probed and can reveal to what extent near-cognate ligands stabilize the folded state to affect gene regulation. Here, we use smFRET to study the effects of preQ0 and guanine in comparison to preQ1 on the kinetics of Bsu aptamer folding. Our preliminary data show that at saturating concentration and in the presence of Mg2+, guanine stabilizes the folded state as potently as preQ0. However, surprisingly, in the absence of Mg2+, guanine tends to stabilize the folded state more efficiently than preQ0. We anticipate this comparative study to elucidate the ligand-mediated folding pathway of the Bsu aptamer.
Keywords: preQ1 riboswitch, Kinetics