Characteristics of the <i>glmS</i> ribozyme suggest only structural roles for divalent metal ions

Roth, Adam; Nahvi, Ali; Lee, Mark N.; Jona, Inbal; Breaker, Ronald R.

doi:10.1261/rna.2266506

Cited by 107 publications

(164 citation statements)

References 63 publications

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“…Early truncation analysis showed that the nucleotides downstream of C75 are nonessential for catalytic activity of the B. subtilis ribozyme , although recent kinetic analyses indicate that a pseudoknot in this region enhances self-cleavage activity by stabilizing the core structure at limiting Mg 2+ concentrations (Wilkinson and Been 2005;Roth et al 2006). For our studies, in which we were particularly interested in the structural impact of ligand binding on the catalytic core, we therefore focused on the structurally presumably more labile catalytic core of the riboswitch.…”

Section: Resultsmentioning

confidence: 99%

“…Data were fit with a single-exponential increase function to yield the reported rate constant k obs . activity of our G1 construct or primarily plays a structural role, as recently suggested for the cis-acting ribozyme (Klein and Ferre-D'Amare 2006;Roth et al 2006). Therefore, we measured cleavage activity under buffer conditions of 50 mM HEPES-KOH (pH 7.5), 200 mM KCl, 2 mM EDTA, and 1 mM Co(NH 3 ) 6 3+ in the presence of 10 mM GlcN6P at 25°C.…”

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confidence: 94%

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Trans-acting glmS catalytic riboswitch: Locked and loaded

Tinsley

Furchak

Walter³

2007

RNA

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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.

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Section: Resultsmentioning

confidence: 99%

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confidence: 94%

Trans-acting glmS catalytic riboswitch: Locked and loaded

Tinsley

Furchak

Walter³

2007

RNA

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“…In certain Gram-positive bacteria, glmS ribozymes repress the production of GlmS protein by monitoring the concentration of its key enzymatic product, GlcN6P. Previously known examples of glmS ribozymes were identified by using comparative sequence analysis algorithms and were exclusively found in Grampositive bacteria (Barrick et al 2004;Winkler et al 2004;Roth et al 2006;Gardner et al 2009). Each bacterium that contains a glmS ribozyme carries a single representative exclusively located in the 59 UTR of its glmS gene.…”

Section: Introductionmentioning

confidence: 99%

An expanded collection and refined consensus model of glmS ribozymes

McCown¹,

Roth²,

Breaker³

2011

RNA

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Self-cleaving glmS ribozymes selectively bind glucosamine-6-phosphate (GlcN6P) and use this metabolite as a cofactor to promote self-cleavage by internal phosphoester transfer. Representatives of the glmS ribozyme class are found in Gram-positive bacteria where they reside in the 59 untranslated regions (UTRs) of glmS messenger RNAs that code for the essential enzyme L-glutamine:D-fructose-6-phosphate aminotransferase. By using comparative sequence analyses, we have expanded the number of glmS ribozyme representatives from 160 to 463. All but two glmS ribozymes are present in glmS mRNAs and most exhibit striking uniformity in sequence and structure, which are features that make representatives attractive targets for antibacterial drug development. However, our discovery of rare variants broadens the consensus sequence and structure model. For example, in the Deinococcus-Thermus phylum, several structural variants exist that carry additional stems within the catalytic core and changes to the architecture of core-supporting substructures. These findings reveal that glmS ribozymes have a broader phylogenetic distribution than previously known and suggest that additional rare structural variants may remain to be discovered.

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“…À5 min À1 to >10 min À1 under physiological conditions upon binding of GlcN6P (McCarthy et al 2005;Roth et al 2006;Klein et al 2007a). On the other hand, glucose-6-phosphate (Glc6P), which has a hydroxyl group instead of an amine group, does not activate self-cleavage of glmS and acts as an inhibitor (McCarthy et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Yao¹,

Hamelberg²

2010

RNA

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The GlmS ribozyme is believed to exploit a general acid-base catalytic mechanism in the presence of glucosamine-6-phosphate (GlcN6P) to accelerate self-cleavage by approximately six orders of magnitude. The general acid and general base are not known, and the role of the GlcN6P cofactor is even less well understood. The amine group of GlcN6P has the ability to either accept or donate a proton and could therefore potentially act as an acid or a base. In order to decipher the role of GlcN6P in the self-cleavage of glmS, we have determined the preferred protonation state of the amine group in the wild-type and an inactive G40A mutant using molecular dynamics simulations and free energy calculations. Here we show that, upon binding of GlcN6P to wild-type glmS, the pK a of the amine moiety is altered by the active site environment, decreasing by about 2.2 from a solution pK a of about 8.2. On the other hand, we show that the pK a of the amine group slightly increases to about 8.4 upon binding to the G40A inactive mutant of glmS. These results suggest that GlcN6P acts as a general acid in the self-cleavage of glmS. Upon binding to glmS, GlcN6P can easily release a proton to the 59-oxygen of G1 during self-cleavage of the backbone phosphodiester bond. However, in the G40A inactive mutant of glmS, the results suggest that the ability of GlcN6P to easily release its proton is diminished, in addition to the possible lack of G40 as an effective base.

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Characteristics of the glmS ribozyme suggest only structural roles for divalent metal ions

Cited by 107 publications

References 63 publications

Trans-acting glmS catalytic riboswitch: Locked and loaded

Trans-acting glmS catalytic riboswitch: Locked and loaded

An expanded collection and refined consensus model of glmS ribozymes

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

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