Ligand Requirements for glmS Ribozyme Self-Cleavage

Abstract

Natural RNA catalysts (ribozymes) perform essential reactions in biological RNA processing and protein synthesis, whereby catalysis is intrinsic to RNA structure alone or in combination with metal ion cofactors. The recently discovered glmS ribozyme is unique in that it functions as a glucosamine-6-phosphate (GlcN6P)-dependent catalyst believed to enable "riboswitch" regulation of amino-sugar biosynthesis in certain prokaryotes. However, it is unclear whether GlcN6P functions as an effector or coenzyme to promote ribozyme self-cleavage. Herein, we demonstrate that ligand is absolutely requisite for glmS ribozyme self-cleavage activity. Furthermore, catalysis both requires and is dependent upon the acid dissociation constant (pKa) of the amine functionality of GlcN6P and related compounds. The data demonstrate that ligand is integral to catalysis, consistent with a coenzyme role for GlcN6P and illustrating an expanded capacity for biological RNA catalysis.

Characteristics of Ligand Recognition by a glmS Self‐Cleaving Ribozyme

“…[1,3,6] Although this K D value is greater than those determined for most other natural riboswitches, glmS ribozymes exhibit a high level of molecular recognition specificity and are able to reject even close chemical analogues of GlcN6P. For example, glucosamine-6-sulphate can induce ribozyme activation to the same extent as GlcN6P, albeit when present at concentrations that are approximately 100-fold greater.…”

Use of a coenzyme by the glmS ribozyme-riboswitch suggests primordial expansion of RNA chemistry by small molecules

“…As expected from the foregoing, the conformation of the Glc6P-bound ribozyme is identical to those of the ribozyme in other functional states [6]. Biochemical studies show that Glc6P competes with GlcN6P for binding to the glmS ribozyme, functioning in effect as an antagonist [12]. Since the ribozyme is pre-folded but completely inactive in the absence of GlcN6P, the most parsimonious conclusion is that this compound functions as a catalytic cofactor of the RNA.…”

“…The functional importance of the amine group of GlcN6P for glmS ribozyme activity is underscored by studies using the isosteric sugar, glucose-6-phosphate (Glc6P). This compound, which differs from GlcN6P only in having a hydroxyl rather than an amine group at position 2 of the pyranose ring, is not an activator of the ribozyme [12]. Cocrystal structures show that it binds precisely in the same location as GlcN6P.…”

“…Conversely, the rigid and pre-folded glmS ribozyme binds GlcN6P in an open, solvent-accessible pocket (figure 1) [5]. Analogue studies [12] underscore the importance of three of the functional groups of GlcN6P that interact with the glmS ribozyme: the anomeric hydroxyl, the amine and the phosphate. A variety of compounds that present vicinal amine and hydroxyl groups are weak activators of the glmS ribozyme.…”

Signal amplification of glucosamine-6-phosphate based on ribozyme glmS

“…Therefore, activators that can be comparable or superior to natural GlcN6P are in great demand for a better understanding of the riboswitch and paving the way to the discovery of effective antibiotics. As the substantial structural and mechanistic information of the glmS riboswitch have been disclosed over recent years, a lot of small molecule agonist and antagonists have been developed and evaluated (Lim et al, 2006;McCarthy et al, 2005;Posakony and Ferre-D'Amare, 2013;Wang et al, 2012). However, the assay for GlcN6P analogues as trigger of the self-cleaving of glmS ribozyme is based on the gel analysis of isotope labelled experiments, which could only be carried out in laboratories with a professional setup.…”