Riboswitches are RNA regulatory elements that govern gene expression by recognition of small molecule ligands via a high affinity aptamer domain. Molecular recognition can lead to active or attenuated gene expression states by controlling accessibility to mRNA signals necessary for transcription or translation. Key areas of inquiry focus on how an aptamer attains specificity for its effector, the extent to which the aptamer folds prior to encountering its ligand, and how ligand binding alters expression signal accessibility. Here we present crystal structures of the preQ 1 riboswitch from Thermoanaerobacter tengcongensis in the preQ 1 -bound and free states. Although the mode of preQ 1 recognition is similar to that observed for preQ 0 , surface plasmon resonance revealed an apparent K D of 2.1 ؎ 0.3 nM for preQ 1 but a value of 35.1 ؎ 6.1 nM for preQ 0 . This difference can be accounted for by interactions between the preQ 1 methylamine and base G5 of the aptamer. To explore conformational states in the absence of metabolite, the free-state aptamer structure was determined. A14 from the ceiling of the ligand pocket shifts into the preQ 1 -binding site, resulting in "closed" access to the metabolite while simultaneously increasing exposure of the ribosome-binding site. Solution scattering data suggest that the free-state aptamer is compact, but the "closed" free-state crystal structure is inadequate to describe the solution scattering data. These observations are distinct from transcriptional preQ 1 riboswitches of the same class that exhibit strictly ligand-dependent folding. Implications for gene regulation are discussed.

The hairpin ribozyme requires functional group contributions from G8 to assist in phosphodiester bond cleavage. Previously, replacement of G8 by a series of nucleobase variants showed little effect on interdomain docking, but a 3-to 250-fold effect on catalysis. To identify G8 features that contribute to catalysis within the hairpin ribozyme active site, structures for five base variants were solved by X-ray crystallography in a resolution range between 2.3 to 2.7 Å. For comparison, a native all-RNA "G8" hairpin ribozyme structure was refined to 2.05 Å resolution. The native structure revealed a scissile bond angle (τ) of 158°, which is close to the requisite 180° 'in-line' geometry. Mutations G8(inosine), G8(diaminopurine), G8(aminopurine), G8(adenosine) and G8(uridine) folded properly, but exhibited non-ideal scissile bond geometries (τ ranging from 118° to 93°) that paralleled their diminished solution activities. A superposition ensemble of all structures, including a previously described hairpin ribozyme-vanadate complex, indicated the scissile bond can adopt a variety of conformations resulting from perturbation of the chemical environment, and provided a rationale for how the exocyclic amine of nucleobase 8 promotes productive, in-line geometry. Changes at position 8 also caused variations in the A−1 sugar pucker. In this regard, variants A8 and U8 appeared to represent non-productive ground-states in which their 2'-OH groups mimicked the pro-R, non-bridging oxygen of the vanadate transition-state complex. Finally, the results indicated that ordered water molecules bind near the 2'-hydroxyl of A−1, lending support to the hypothesis that solvent may play an important role in the reaction. † This work was supported by NIH Grant GM63162 to J.E.W. ‡ Protein Data Bank Codes for the reported structures: 1ZFR (G8), 1ZFT (G8I), 1ZFV (G8A), 1ZFX (G8U), 2BCY (G8AP), 2BB1 (G8DAP), 2BCZ (G8I/dA−1). § Present address: Rosalind Franklin School of Science and Medicine, Dept. Biochem. and Mol. Biol., 3333 Green Bay Road Rd., N. Chicago, IL 60064, USA * To whom correspondence should be addressed: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Box 712, Rochester, New York 14642 USA. Phone: 585 273-4516; Fax: 585 275-6007; Email: Joseph_Wedekind@URMC.Rochester.edu ∥ These authors contributed equally to this work.Supporting Information Available A stereo diagram of representative electron density maps is provided for the native and position 8 variants of this study (Figure 1). A diagram comparing the G8I/2'-OMe A−1 and G8I/2'-deoxy A−1 variants is also available (Figure 2). The method for HPLC composition analysis of AP8 and DAP8 crystals is reported, as well as elution profiles for the separated hairpin ribozyme strands (Figure 3). This information is provided free of charge at http://pubs.acs.org NIH Public Access The hairpin ribozyme is a small ribozyme whose family members catalyze a reversible, sitespecific phosphodiester bond cleavage reacti...

The potential for water to participate in RNA catalyzed reactions has been the topic of several recent studies. Here, we report crystals of a minimal, hinged hairpin ribozyme in complex with the transition-state analog vanadate at 2.05 Å resolution. Waters are present in the active site and are discussed in light of existing views of catalytic strategies employed by the hairpin ribozyme. A second structure harboring a 29,59-phosphodiester linkage at the site of cleavage was also solved at 2.35 Å resolution and corroborates the assignment of active site waters in the structure containing vanadate. A comparison of the two structures reveals that the 29,59 structure adopts a conformation that resembles the reaction intermediate in terms of (1) the positioning of its nonbridging oxygens and (2) the covalent attachment of the 29-O nucleophile with the scissile G+1 phosphorus. The 29,59-linked structure was then overlaid with scissile bonds of other small ribozymes including the glmS metabolite-sensing riboswitch and the hammerhead ribozyme, and suggests the potential of the 29,59 linkage to elicit a reaction-intermediate conformation without the need to form metalloenzyme complexes. The hairpin ribozyme structures presented here also suggest how water molecules bound at each of the nonbridging oxygens of G+1 may electrostatically stabilize the transition state in a manner that supplements nucleobase functional groups. Such coordination has not been reported for small ribozymes, but is consistent with the structures of protein enzymes. Overall, this work establishes significant parallels between the RNA and protein enzyme worlds.Keywords: hairpin ribozyme; 29,59-phosphodiester; vanadate; reaction intermediate; transition-state stabilization; active-site waters INTRODUCTIONProtein enzymes have evolved numerous strategies to lower the energetic barrier required to convert substrates into products (Knowles 1991). Polanyi and Pauling perceptively envisioned that this could be accomplished by distorting the precatalytic geometry of the reactants into that of the transition state, which entails the expenditure of binding energy derived from the substrate-protein interaction (Lienhard 1973;Borman and Wolfenden 2004). As such, knowledge of the stereochemical interactions employed by an enzyme during the transition state can provide great insight into the chemical strategies utilized by the catalyst to accelerate a reaction rate (Lienhard 1973). Although envisioning the transition state of a phosphoryl-transfer reaction would appear somewhat trivial (Dennis and Westheimer 1966;Knowles 1980), understanding the factors leading to formation and stabilization of such intermediates for a given enzyme is not. This statement is especially true in the case of RNA enzymes, whose rateenhancing features are only beginning to be understood (Doherty and Doudna 2001;Doudna and Lorsch 2005;Fedor and Williamson 2005;Bevilacqua and Yajima 2006), despite the conserved and essential nature of ribocatalysts in biology.Although it is diffi...

PreQ1 riboswitches regulate genes by binding the pyrrolopyrimidine intermediate preQ1 during biosynthesis of the essential tRNA base queuosine. We report the first preQ1-II riboswitch structure at 2.3 Å resolution, which uses a novel fold to achieve effector recognition at the confluence of a three-way-helical junction flanking a pseudoknotted ribosome-binding site (RBS). The results account for preQ1-II-riboswitch-mediated translational control, and expand the known repertoire of ligand binding modes utilized by regulatory RNAs.

Human APOBEC3G (hA3G) is a cytidine deaminase that restricts human immunodeficiency virus (HIV)-1 infection in a vif(the virion infectivity factor from HIV)-dependent manner. hA3G from HIV-permissive activated CD4؉ T-cells exists as an inactive, high molecular mass (HMM) complex that can be transformed in vitro into an active, low molecular mass (LMM) variant comparable with that of HIV-non-permissive CD4؉ T-cells. Here we present low resolution structures of hA3G in HMM and LMM forms determined by small angle x-ray scattering and advanced shape reconstruction methods. The results show that LMM particles have an extended shape, dissimilar to known cytidine deaminases, featuring novel tail-to-tail dimerization. Shape analysis of LMM and HMM structures revealed how symmetric association of dimers could lead to minimal HMM variants. These observations imply that the disruption of cellular HMM particles may require regulation of protein-RNA, as well as protein-protein interactions, which has implications for therapeutic development.

Riboswitches are RNA elements that control gene expression through metabolite binding. The preQ 1 riboswitch exhibits the smallest known ligand-binding domain and is of interest for its economical organization and high affinity interactions with guanine-derived metabolites required to confer tRNA wobbling. Here we present the crystal structure of a preQ 1 aptamer domain in complex with its precursor metabolite preQ 0 . The structure is highly compact with a core that features a stem capped by a well organized decaloop. The metabolite is recognized within a deep pocket via Watson-Crick pairing with C15. Additional hydrogen bonds are made to invariant bases U6 and A29. The ligand-bound state confers continuous helical stacking throughout the core fold, thus providing a platform to promote Watson-Crick base pairing between C9 of the decaloop and the first base of the ribosome-binding site, G33. The structure offers insight into the mode of ribosome-binding site sequestration by a minimal RNA fold stabilized by metabolite binding and has implications for understanding the molecular basis by which bacterial genes are regulated.Riboswitches are naturally occurring, structured motifs in the 5Ј-untranslated regions of a handful of mRNAs. It has been estimated that these elements regulate the expression of 3-4% of bacterial genes (1). Their mechanism of action entails "sensing" a cellular metabolite via a high affinity aptamer domain, which alters the accessibility of flanking mRNA sequences necessary for control of transcription or translation (2, 3). Respective riboswitches have been discovered that sense more than a dozen distinct small molecules (reviewed in Ref. 4), and these RNA-regulatory elements have been identified in the genomes of several human pathogens (5-7). As such, elucidating the principles by which riboswitches bind their cognate ligands is critical for the identification and validation of new antibiotic targets (8, 9).Queuosine (Q) 5 is a hypermodified variant of guanosine necessary for wobbling of certain tRNAs (10). This modification improves translational accuracy (11-13) and pervades both prokaryotic and eukaryotic phyla. De novo Q synthesis occurs only in bacteria, requiring that humans acquire it from gut flora or dietary sources (14). Production of Q begins with GTP and proceeds via formation of the metabolic intermediate preQ 0 (see Fig. 1A, inset), the antecedent to preQ 1 (15). Breaker and co-workers (16) discovered recently that some genes encoding proteins whose function is preQ 1 uptake or biosynthesis are regulated by riboswitches responsive to this metabolite and its analogs. Equilibrium dialysis revealed that a representative riboswitch aptamer favors preQ 1 binding over preQ 0 by only 5-fold, with preQ 0 displaying a K d of ϳ100 nM (16). Phylogenetic comparisons suggested a stem-loop secondary structure (see Fig. 1A) that could be divided further into two aptamer "types" based on sequence differences in the L1 region.In contrast to other riboswitches, the preQ 1 aptamer is unusually s...

The hairpin ribozyme is an RNA enzyme that performs site-specific phosphodiester bond cleavage between nucleotides A-1 and G+1 within its cognate substrate. Previous functional studies revealed that the minimal hairpin ribozyme exhibited "gain-of-function" cleavage properties resulting from U39C or U39 to propyl linker (C3) modifications. Furthermore, each "mutant" displayed different magnesium-dependence in its activity. To investigate the molecular basis for these gain-of-function variants, crystal structures of minimal, junctionless hairpin ribozymes were solved in native (U39), and mutant U39C and U39(C3) forms. The results revealed an overall molecular architecture comprising two docked internal loop domains folded into a wishbone shape, whose tertiary interface forms a sequestered active site. All three minimal hairpin ribozymes bound Co(NH(3))(6)(3+) at G21/A40, the E-loop/S-turn boundary. The native structure also showed that U37 of the S-turn adopts both sequestered and exposed conformations that differ by a maximum displacement of 13 A. In the sequestered form, the U37 base packs against G36, and its 2'-hydroxyl group forms a water mediated hydrogen bond to O4' of G+1. These interactions were not observed in previous four-way-junction hairpin ribozyme structures due to crystal contacts with the U1A splicing protein. Interestingly, the U39C and U39(C3) mutations shifted the equilibrium conformation of U37 into the sequestered form through formation of new hydrogen bonds in the S-turn, proximal to the essential nucleotide A38. A comparison of all three new structures has implications for the catalytically relevant conformation of the S-turn and suggests a rationale for the distinctive metal dependence of each mutant.

Catalytic RNA molecules can achieve rate acceleration by shifting base pK(a) values toward neutrality. Prior evidence has suggested that base A38 of the hairpin ribozyme plays an important role in phosphoryl transfer, possibly functioning as a general acid, or by orienting a specific water molecule for proton transfer. To address the role of A38, we used Raman spectroscopy to measure directly the pK(a) of the N1-imino moiety in the context of hairpin ribozyme crystals representative of a "precatalytic" conformation. The results revealed that the pK(a) of A38 is shifted to 5.46 +/- 0.05 relative to 3.68 +/- 0.06 derived from a reference solution of the nucleotide AMP. The elevated pK(a) correlates well with the first titration point of the macroscopic pH-rate profile of the hairpin ribozyme in solution and strongly supports A38 as a general acid catalyst in bond scission. The results confirm that A38 is protonated before the transition state, which would promote phosphorane development. Overall, the results establish a cogent structure-function paradigm that expands our understanding of how RNA structure can enhance nucleobase reactivity to catalyze biological reactions.