Trans-acting glmS catalytic riboswitch: Locked and loaded

Abstract

A recently discovered class of gene regulatory RNAs, coined riboswitches, are commonly found in noncoding segments of bacterial and some eukaryotic mRNAs. Gene up-or down-regulation is triggered by binding of a small organic metabolite, which typically induces an RNA conformational change. Unique among these noncoding RNAs is the glmS catalytic riboswitch, or ribozyme, found in the 59-untranslated region of the glmS gene in Gram-positive bacteria. It is activated by glucosamine-6-phosphate (GlcN6P), leading to site-specific backbone cleavage of the mRNA and subsequent repression of the glmS gene, responsible for cellular GlcN6P production. Recent biochemical and structural evidence suggests that the GlcN6P ligand acts as a coenzyme and participates in the cleavage reaction without inducing a conformational change. To better understand the role of GlcN6P in solution structural dynamics and function, we have separated the glmS riboswitch core from Bacillus subtilis into a trans-cleaving ribozyme and an externally cleaved substrate. We find that trans cleavage is rapidly activated by nearly 5000-fold to a rate of 4.4 min À1 upon addition of 10 mM GlcN6P, comparable to the cis-acting ribozyme. Fluorescence resonance energy transfer suggests that this ribozyme-substrate complex does not undergo a global conformational change upon ligand binding in solution. In addition, footprinting at nucleotide resolution using terbium(III) and RNase V1 indicates no significant changes in secondary and tertiary structure upon ligand binding. These findings suggest that the glmS ribozyme is fully folded in solution prior to binding its activating ligand, supporting recent observations in the crystalline state.

RNA dynamics: it is about time

“…For example, NMR studies show that an RNA thermosensor regulates expression of heat/cold shock genes by progressively undergoing conformational changes against a temperature gradient [17•] (Figure 3e). By contrast, the aptamer domain of the glmS catalytic riboswitch binds its target glucosamine-6-phosphate (GlcN6P) without undergoing a significant conformational change [18]. Here, GlcN6P acts as a co-enzyme in the cleavage reaction, and its binding contributes the missing chemical participants for selfcleavage as the signal that leads to mRNA degradation.…”

“…Single molecule FRET also allowed for the dissection of the metal-ion dependent multi-step folding pathways of an in vitro selected Diels-Alderase ribozyme [26] and a DNAzyme [27]. Sometimes FRET can demonstrate the lack of significant conformational dynamics of a ribozyme, for example of the glmS ribozyme upon cofactor binding [18]. Often a specific tertiary interaction of a larger ribozyme can be studied in isolation, which led, for example, to the FRET-based characterization of docking and undocking of the GAAA tetraloop and receptor as induced by metal ions and increased hydrostatic pressure, respectively [28,29].…”

Structural features of metabolite-sensing riboswitches

“…For this reason, multiple structures of glmS ribozymes were able to be determined in the presence or absence of natural ligands or potential inhibitors. These studies combined with subsequent fluorescence resonance energy transfer (FRET) experiments suggest that GlcN6P acts an enzyme cofactor [52], providing the final piece to a preorganized nucleobase active site. Elucidating the catalytic contribution of each component of the glmS active site is being intensely studied and future work should provide additional insight into this remarkable genetic regulatory ribozyme.…”

Non-nearest-neighbor dependence of the stability for RNA group II single-nucleotide bulge loops

“…Among these are the control of gene expression (Chung et al 2006), intron splicing (Schmelzer and Schweyen 1986;Suzuki et al 2006), protein synthesis and catalysis (Doudna et al 1989;Krasovska et al 2006), ligand binding (Harper and Logsdon 1991), and virus replication (Roy et al 1990). Recent discoveries have highlighted the regulation of gene expression by microRNAs (miRNAs) (Matzke and Birchler 2005) and riboswitches (Tinsley et al 2007). Given its versatility in a wide variety of biological functions, RNA has been recognized as much more than a passive intermediary between DNA and proteins.…”