Trans-acting glmS catalytic riboswitch: locked and loaded

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 5'-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(-1) 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.     

Rapid steps in the glmS ribozyme catalytic pathway: cation and ligand requirements

The glmS ribozyme is a conserved riboswitch found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine 6-phosphate (GlcN6P). GlcN6P binding promotes site-specific self-cleavage in the 5' UTR of the glmS mRNA, resulting in downregulation of gene expression. The glmS ribozyme has previously been shown to lack strong cation specificity when the rate-limiting folding step of the cleavage reaction pathway is measured. This does not provide data regarding cation and ligand specificities of the glmS ribozyme during the rapid ligand binding chemical catalysis events. Prefolding of the ribozyme in Mg(2+)-containing buffers effectively isolates the rapid ligand binding and catalytic events (k(obs) > 60 min(-1)) from rate-limiting folding (k(obs) < 4 min(-1)). Here we employ this experimental design to assay the cations and ligand requirements for rapid ligand binding and catalysis. We show that molar concentrations of monovalent cations are also capable of inducing the formation of the native GlcN6P binding structure but are unable to promote ligand binding and catalysis rates of >4 min(-1). Our data show that the sole obligatory role for divalent cations, for which there is crystallographic evidence, is coordination of the phosphate moiety of GlcN6P in the ligand-binding pocket. In further support of this hypothesis, our data show that a nonphosphorylated analogue of GlcN6P, glucosamine, is unable to promote rapid ligand binding and catalysis in the presence of divalent cations. Folding of the ribozyme is, therefore, relatively independent of cation identity, but the rapid initiation of catalysis upon the addition of ligand is stricter.     

Requirement of helix P2.2 and nucleotide G1 for positioning the cleavage site and cofactor of the glmS ribozyme

The glmS ribozyme is a catalytic RNA that self-cleaves at its 5'-end in the presence of glucosamine 6-phosphate (GlcN6P). We present structures of the glmS ribozyme from Thermoanaerobacter tengcongensis that are bound with the cofactor GlcN6P or the inhibitor glucose 6-phosphate (Glc6P) at 1.7 A and 2.2 A resolution, respectively. The two structures are indistinguishable in the conformations of the small molecules and of the RNA. GlcN6P binding becomes apparent crystallographically when the pH is raised to 8.5, where the ribozyme conformation is identical with that observed previously at pH 5.5. A key structural feature of this ribozyme is a short duplex (P2.2) that is formed between sequences just 3' of the cleavage site and within the core domain, and which introduces a pseudoknot into the active site. Mutagenesis indicates that P2.2 is required for activity in cis-acting and trans-acting forms of the ribozyme. P2.2 formation in a trans-acting ribozyme was exploited to demonstrate that N1 of the guanine at position 1 contributes to GlcN6P binding by interacting with the phosphate of the cofactor. At neutral pH, RNAs with adenine, 2-aminopurine, dimethyladenine or purine substitutions at position 1 cleave faster with glucosamine than with GlcN6P. This altered cofactor preference provides biochemical support for the orientation of the cofactor within the active site. Our results establish two features of the glmS ribozyme that are important for its activity: a sequence within the core domain that selects and positions the cleavage-site sequence, and a nucleobase at position 1 that helps position GlcN6P.     

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

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 5'-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.     

Evidence for preorganization of the glmS ribozyme ligand binding pocket

We have examined the tertiary structure of the ligand-activated glmS ribozyme by a combination of methods with the aim of evaluating the magnitude of RNA conformational change induced by binding of the cofactor, glucosamine 6-phosphate (GlcN6P). Hydroxyl radical footprinting of a trans-acting ribozyme complex identifies several sites of solvent protection upon incubation of the RNA in Mg(2+)-containing solutions, providing initial evidence of the tertiary fold of the ribozyme. Under these folding conditions and at GlcN6P concentrations that saturate the ligand-induced cleavage reaction, we do not observe changes to this pattern. Cross-linking with short-wave UV light of the complex yielded similar overall results. In addition, ribozyme-substrate complexes cross-linked in the absence of GlcN6P could be gel purified and then activated in the presence of ligand. One of these active cross-linked species links the base immediately 3' of the cleavage site to a highly conserved region of the ribozyme core and could be catalytically activated by ligand. Combined with recent studies that argue that GlcN6P acts as a coenzyme in the reaction, our data point to a riboswitch mechanism in which ligand binds to a prefolded active site pocket and assists in catalysis via a direct participation in the reaction chemistry, the local influence on the geometry of the active site constituents, or a combination of both mechanisms. This mode of action is different from that observed for other riboswitches characterized to date, which act by inducing secondary and tertiary structure changes.     

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

The glmS ribozyme-riboswitch is the first known example of a naturally occurring catalytic RNA that employs a small molecule as a coenzyme. Binding of glucosamine-6-phosphate (GlcN6P) activates self-cleavage of the bacterial ribozyme, which is part of the mRNA encoding the metabolic enzyme GlcN6P-synthetase. Cleavage leads to negative feedback regulation. GlcN6P binds in the active site of the ribozyme, where its amine could function as a general acid and electrostatic catalyst. The ribozyme is pre-folded but inactive in the absence of GlcN6P, demonstrating it has evolved strict dependence on the exogenous small molecule. The ribozyme showcases the ability of RNA to co-opt non-covalently bound small molecules to expand its chemical repertoire. Analogue studies demonstrate that some molecules other than GlcN6P, such as l-serine (but not d-serine), can function as weak activators. This suggests how coenzyme use by RNA world ribozymes may have led to evolution of proteins. Primordial cofactor-dependent ribozymes may have evolved to bind their cofactors covalently. If amino acids were used as cofactors, this could have driven the evolution of RNA aminoacylation. The ability to make covalently bound peptide coenzymes may have further increased the fitness of such primordial ribozymes, providing a selective pressure for the invention of translation.     

Examination of the structural and functional versatility of glmS ribozymes by using in vitro selection

Self-cleaving ribozymes associated with the glmS genes of many Gram-positive bacteria are activated by binding to glucosamine-6-phosphate (GlcN6P). Representatives of the glmS ribozyme class function as metabolite-sensing riboswitches whose self-cleavage activities down-regulate the expression of GlmS enzymes that synthesizes GlcN6P. As with other riboswitches, natural glmS ribozyme isolates are highly specific for their target metabolite. Other small molecules closely related to GlcN6P, such as glucose-6-phosphate, cannot activate self-cleavage. We applied in vitro selection methods in an attempt to identify variants of a Bacillus cereus glmS ribozyme that expand the range of compounds that induce self-cleavage. In addition, we sought to increase the number of variant ribozymes of this class to further examine the proposed secondary structure model. Although numerous variant ribozymes were obtained that efficiently self-cleave, none exhibited changes in target specificity. These findings are consistent with the hypothesis that GlcN6P is used by the ribozyme as a coenzyme for RNA cleavage, rather than an allosteric effector.     

An expanded collection and refined consensus model of glmS ribozymes

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 5' 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.     

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

The glmS ribozyme is a riboswitch class that occurs in certain Gram-positive bacteria, where it resides within mRNAs encoding glucosamine 6-phosphate synthase. Members of this self-cleaving ribozyme class rapidly catalyze RNA transesterification upon binding GlcN6P, and genetic evidence suggests that this cleavage event is important for down-regulating GlmS protein expression. In this report, we present a refined secondary structure model of the glmS ribozyme and determine the importance of a conserved pseudoknot structure for optimal ribozyme function. Analyses of deletion constructs demonstrate that the pseudoknot, together with other structural elements, permits the ribozyme to achieve maximum rate constants for RNA cleavage at physiologically relevant Mg2+ concentrations. In addition, we show that substantial rate enhancements are supported by an exchange-inert cobalt (III) complex and by molar concentrations of monovalent ions. Our findings indicate that the glmS ribozyme forms a complex structure to employ catalytic strategies that do not require the direct participation of divalent metal ions.     

Core requirements for glmS ribozyme self-cleavage reveal a putative pseudoknot structure

The glmS ribozyme is a self-cleaving RNA catalyst that resides in the 5′-untranslated region of glmS mRNA in certain bacteria. The ribozyme is specifically activated by glucosamine-6-phosphate (GlcN6P), the metabolic product of the GlmS protein, and is thus proposed to provide a feedback mechanism of riboswitch regulation. Both phylogenetic and biochemical analyses of the glmS ribozyme have established a highly conserved core sequence and secondary structure required for GlcN6P-dependent self-cleavage. However, the high degree of nucleotide conservation offers few clues regarding the higher-order structural organization of the catalytic core. To further investigate core ribozyme structure, minimal ‘consensus-type’ glmS ribozymes that retain GlcN6P-dependent activity were produced. Mutational analyses of consensus-type glmS ribozymes support a model for core ribozyme folding through a pseudoknot structure formed by the interaction of two highly conserved sequence segments. Moreover, GlcN6P-dependent function is demonstrated for bimolecular constructs in which substrate interaction with the ribozyme is minimally comprised of sequence representing that involved in putative pseudoknot formation. These studies suggest that the glmS ribozyme adopts an intricate multi-strand catalytic core through the formation of a pseudoknot structure, and provide a refined model for further considering GlcN6P interaction and GlcN6P-dependent ribozyme function.     

Caged glucosamine-6-phosphate for the light-control of riboswitch activity

We have synthesized a light-activatable ("caged") derivative of glucosamine-6-phosphate (GlcN6P), which only upon irradiation becomes a cofactor for the glmS riboswitch. This glmS riboswitch maintains its activity when embedded in the 3'-untranslated region of eukaryotic mRNA molecules and caged GlcN6P reduces the amount of translated EGFP upon irradiation with light in vitro.     

NAIM and site-specific functional group modification analysis of RNase P RNA: magnesium dependent structure within the conserved P1-P4 multihelix junction contributes to catalysis

The tRNA processing endonuclease ribonuclease P contains an essential and highly conserved RNA molecule (RNase P RNA) that is the catalytic subunit of the enzyme. To identify and characterize functional groups involved in RNase P RNA catalysis, we applied self-cleaving ribozyme-substrate conjugates, on the basis of the RNase P RNA from Escherichia coli, in nucleotide analogue interference mapping (NAIM) and site-specific modification experiments. At high monovalent ion concentrations (3 M) that facilitate protein-independent substrate binding, we find that the ribozyme is largely insensitive to analogue substitution and that concentrations of Mg2+ (1.25 mM) well below that necessary for optimal catalytic rate (>100 mM) are required to produce interference effects because of modification of nucleotide bases. An examination of the pH dependence of the reaction rate at 1.25 mM Mg2+ indicates that the increased sensitivity to analogue interference is not due to a change in the rate-limiting step. The nucleotide positions detected by NAIM under these conditions are located exclusively in the catalytic domain, consistent with the proposed global structure of the ribozyme, and predominantly occur within the highly conserved P1-P4 multihelix junction. Several sensitive positions in J3/4 and J2/4 are proximal to a previously identified site of divalent metal ion binding in the P1-P4 element. Kinetic analysis of ribozymes with site-specific N7-deazaadenosine and deazaguanosine modifications in J3/4 was, in general, consistent with the interference results and also permitted the analysis of sites not accessible by NAIM. These results show that, in this region only, modification of the N7 positions of A62, A65, and A66 resulted in measurable effects on reaction rate and modification at each position displayed distinct sensitivities to Mg2+ concentration. These results reveal a restricted subset of individual functional groups within the catalytic domain that are particularly important for substrate cleavage and demonstrate a close association between catalytic function and metal ion-dependent structure in the highly conserved P1-P4 multihelix junction.     

Investigation of adenosine base ionization in the hairpin ribozyme by nucleotide analog interference mapping.

Tertiary structure in globular RNA folds can create local environments that lead to pKa perturbation of specific nucleotide functional groups. To assess the prevalence of functionally relevant adenosine-specific pKa perturbation in RNA structure, we have altered the nucleotide analog interference mapping (NAIM) approach to include a series of a phosphorothioate-tagged adenosine analogs with shifted N1 pKa values. We have used these analogs to analyze the hairpin ribozyme, a small self-cleaving/ligating RNA catalyst that is proposed to employ a general acid-base reaction mechanism. A single adenosine (A10) within the ribozyme active site displayed an interference pattern consistent with a functionally significant base ionization. The exocyclic amino group of a second adenosine (A38) contributes substantially to hairpin catalysis, but ionization of the nucleotide does not appear to be important for activity. Within the hairpin ribozyme crystal structure, A10 and A38 line opposite edges of a solvent-excluded cavity adjacent to the 5'-OH nucleophile. The results are inconsistent with the model of ribozyme chemistry in which A38 acts as a general acid-base catalyst, and suggest that the hairpin ribozyme uses an alternative mechanism to achieve catalytic rate enhancement that utilizes functional groups within a solvent-excluded cleft in the ribozyme active site.     

Co-ordinated regulation of amino sugar biosynthesis and degradation: the NagC repressor acts as both an activator and a repressor for the transcription of the glmUS operon and requires two separated NagC binding sites.

The NagC repressor controls the expression of the divergently transcribed nagE-nagBACD operons involved in the uptake and degradation of the amino sugars, N-acetyl-D-glucosamine (GlcNAc) and glucosamine (GlcN). The glmUS operon, encoding proteins necessary for the synthesis of GlcN (glmS) and the formation of UDP-GlcNAc (glmU), is transcribed from two promoters located upstream of glmU. In the absence of amino sugars both promoters are active. However, in the presence of GlcNAc, the glmU proximal promoter, P1, is inactive while the upstream promoter, P2, is subject to weak induction. Two binding sites for the NagC repressor are located at -200 and -47 bp upstream of P1. Mutations which prevent NagC binding to either of these sites eliminate expression from the P1 promoter. This shows that binding of NagC is necessary for expression of the glmU P1 promoter and implies that NagC is playing the role of activator for this promoter. Moreover, the location of the distal NagC site suggests that this site is behaving like an upstream activating sequence (UAS).     

Delta ribozyme has the ability to cleave in transan mRNA.

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy.     

Comparative analysis of hairpin ribozyme structures and interference data

Great strides in understanding the molecular underpinnings of RNA catalysis have been achieved with advances in RNA structure determination by NMR spectroscopy and X-ray crystallography. Despite these successes the functional relevance of a given structure can only be assessed upon comparison with biochemical studies performed on functioning RNA molecules. The hairpin ribozyme presents an excellent case study for such a comparison. The active site is comprised of two stems each with an internal loop that forms a series of non-canonical base pairs. These loops dock into each other to create an active site for catalysis. Recently, three independent structures have been determined for this catalytic RNA, including two NMR structures of the isolated loop A and loop B stems and a high-resolution crystal structure of both loops in a docked conformation. These structures differ significantly both in their tertiary fold and the nature of the non-canonical base pairs formed within each loop. Several of the chemical groups required to achieve a functioning hairpin ribozyme have been determined by nucleotide analog interference mapping (NAIM). Here we compare the three hairpin structures with previously published NAIM data to assess the convergence between the structural and functional data. While there is significant disparity between the interference data and the individual NMR loop structures, there is almost complete congruity with the X-ray structure. The only significant differences cluster around an occluded pocket adjacent to the scissile phosphate. These local differences may suggest a role for these atoms in the transition state, either directly in chemistry or via a local structural rearrangement.     

The Varkud satellite ribozyme

The VS ribozyme is the largest nucleolytic ribozyme, for which there is no crystal structure to date. The ribozyme consists of five helical sections, organized by two three-way junctions. The global structure has been determined by solution methods, particularly FRET. The substrate stem-loop binds into a cleft formed between two helices, while making a loop-loop contact with another section of the ribozyme. The scissile phosphate makes a close contact with an internal loop (the A730 loop), the probable active site of the ribozyme. This loop contains a particularly critical nucleotide A756. Most changes to this nucleotide lead to three-orders of magnitude slower cleavage, and the Watson-Crick edge is especially important. NAIM experiments indicate that a protonated base is required at this position for the ligation reaction. A756 is thus a strong candidate for nucleobase participation in the catalytic chemistry.     

Ionization of a critical adenosine residue in the neurospora Varkud Satellite ribozyme active site

The Varkud Satellite (VS) ribozyme catalyzes a site-specific self-cleavage reaction that generates 5'-OH and 2',3'-cyclic phosphate products. Other ribozymes that perform an equivalent reaction appear to employ ionization of an active site residue, either to neutralize the negatively charged transition state or to act as a general acid-base catalyst. To test for important base ionization events in the VS ribozyme ligation reaction, we performed nucleotide analogue interference mapping (NAIM) with a series of ionization-sensitive adenosine and cytidine analogues. A756, a catalytically critical residue located within the VS active site, was the only nucleotide throughout the VS ribozyme that displayed the pH-dependent interference pattern characteristic of functional base ionization. We observed unique rescue of 8-azaadenosine (pK(a) 2.2) and purine riboside (pK(a) 2.1) interference at A756 at reduced reaction pH, suggestive of an ionization-specific effect. These results are consistent with protonation and/or deprotonation of A756 playing a direct role in the VS ribozyme reaction mechanism. In addition, NAIM experiments identified several functional groups within the RNA that play important roles in ribozyme folding and/or catalysis. These include residues in helix II, helix VI (730 loop), the II-III-VI and III-IV-V helix junctions, and loop V.