Folding and Catalysis of the glmS Ribozyme Riboswitch Studied at the Single-Molecule Level

Abstract

We present findings from optical trapping experiments performed on the glmS ribozyme riboswitch. Found in the glmS gene in many bacteria, the glmS riboswitch down-regulates GlmS expression in the presence of the cell wall precursor—and enzymatic product of GlmS—glucosamine-6-phosphate (GlcN6P). In response to GlcN6P, the riboswitch site-specifically cleaves itself near its 5ʹ end, which targets the mRNA for subsequent degradation1. We performed single-molecule force spectroscopy experiments on the ribozyme catalytic core, unfolding and refolding the glmS RNA under controlled mechanical loads. The force-extension curves (FECs) reveal that folding and unfolding occur through a series of intermediate states, and we observe a striking hysteresis between unfolding and refolding of the ribozyme. Analysis of the FEC data leads to a model for the major unfolding pathway, involving the sequential unfolding of a series of discrete substructures. We’ve also begun to characterize the folding energy landscape of the catalytic core, using a series of constant-force measurements. We performed self-cleavage assays with optically trapped ribozyme molecules in the presence of the enzymatic cofactor GlcN6P and its catalytically inactive analog, glucose 6-phosphate. These assays demonstrate that our experimental construct, consisting of aminimal ribozyme sequence, is enzymatically active and strictly dependent upon the presence of GlcN6P. Self-cleavage measurements under controlled loads allow us to determine the profile of the catalytic activity in response to destabilizing forces. We observe an intermediate destabilization regime where the ribozyme remains active, but displays a partial loss of function.Footnotes1 Collins, J.A., Irnov, I., Baker, S., and Winkler, W.C. (2007) Genes Dev21(24), 3356-68.