The distributions,mechanisms, and structures of metabolite-binding riboswitches

Background  

Riboswitches are noncoding RNA structures that appropriately regulate genes in response to changing cellular conditions. The expression of many proteins involved in fundamental metabolic processes is controlled by riboswitches that sense relevant small molecule ligands. Metabolite-binding riboswitches that recognize adenosylcobalamin (AdoCbl), thiamin pyrophosphate (TPP), lysine, glycine, flavin mononucleotide (FMN), guanine, adenine, glucosamine-6-phosphate (GlcN6P), 7-aminoethyl 7-deazaguanine (preQ₁), and S-adenosylmethionine (SAM) have been reported.     

The catalytic diversity of RNAs

The natural RNA enzymes catalyse phosphate-group transfer and peptide-bond formation. Initially, metal ions were proposed to supply the chemical versatility that nucleotides lack. In the ensuing decades, structural and mechanistic studies have substantially altered this initial viewpoint. Whereas self-splicing ribozymes clearly rely on essential metal-ion cofactors, self-cleaving ribozymes seem to use nucleotide bases for their catalytic chemistry. Despite the overall differences in chemical features, both RNA and protein enzymes use similar catalytic strategies.     

Metal ions in the structure and function of RNA

It is becoming increasingly clear that RNA is more than a passive carrier of genetic information. Folded RNA molecules play key roles in almost every aspect of cellular metabolism, including protein transport, RNA splicing, peptide bond formation, and translational regulation. This is facilitated by the multifunctional nature of RNA biopolymers which can serve as rigid structural scaffolds, conformational switches, and catalysts for chemical reactions. In all cases, metal ions play a crucial role in RNA function. For folded RNA molecules, the pathway for adopting proper tertiary structure, and the stabilization of that structure, depends on specific and nonspecific interactions with certain classes of metal ions. There is a rapidly expanding repertoire of RNA structural motifs that typically sequester metal ions, and these are being studied using new spectroscopic and chemical methodologies. Many ribozymes (catalytic RNA molecules) depend on metal ions as cofactors that are explicitly involved in the chemical mechanism of catalysis. All of these functions are exemplified by recent studies of group II introns, which are among the largest ribozymes found in Nature. In this case, there are specific roles for metal ions in the folding pathway, the tertiary structure and the chemical mechanism.     

The chemical versatility of RNA

The ability of RNA to both store genetic information and catalyse chemical reactions has led to the hypothesis that it predates DNA and proteins. While there is no doubt that RNA is capable of storing the genetic information of a primitive organism, only two classes of reactions-phosphoryl transfer and peptide bond formation-have been observed to be catalysed by RNA in nature. However, these naturally occurring ribozymes use a wide range of catalytic strategies that could be applied to other reactions. Furthermore, RNA can bind several cofactors that are used by protein enzymes to facilitate a wide variety of chemical processes. Despite its limited functional groups, these observations indicate RNA is a versatile molecule that could, in principle, catalyse the myriad reactions necessary to sustain life.     

Design of allosteric hammerhead ribozymes activated by ligand-induced structure stabilization.

BACKGROUND: Ribozymes can function as allosteric enzymes that undergo a conformational change upon ligand binding to a site other than the active site. Although allosteric ribozymes are not known to exist in nature, nucleic acids appear to be well suited to display such advanced forms of kinetic control. Current research explores the mechanisms of allosteric ribozymes as well as the strategies and methods that can be used to create new controllable enzymes. RESULTS: In this study, we exploit the modular nature of certain functional RNAs to engineer allosteric ribozymes that are activated by flavin mononucleotide (FMN) or theophylline. By joining an FMN- or theophylline-binding domain to a hammerhead ribozyme by different stem II elements, we have identified a minimal connective bridge comprised of a G.U wobble pair that is responsive to ligand binding. Binding of FMN or theophylline to its allosteric site induces a conformational change in the RNA that stabilizes the wobble pair and ultimately favors the active form of the catalytic core. These ligand-sensitive ribozymes exhibit rate enhancements of more than 100-fold in the presence of FMN and of approximately 40-fold in the presence of theophylline. CONCLUSIONS: An adaptive strategy for modular rational design has proven to be an effective approach to the engineering of novel allosteric ribozymes. This strategy was used to create allosteric ribozymes that function by a mechanism involving ligand-induced structure stabilization. Conceivably, similar engineering strategies and allosteric mechanisms could be used to create a variety of novel allosteric ribozymes that function with other effector molecules.     

Flavin recognition by an RNA aptamer targeted toward FAD

Flavin adenine dinucleotide (FAD) is one of the primary cofactors in biological redox reactions. Designing cofactor-dependent redox ribozymes could benefit from studies of new RNA-cofactor complexes, as would our understanding of ribozyme evolution during an RNA World. We have therefore used the SELEX method to identify RNA aptamers that recognize FAD. Functional analysis of mutant aptamers, S1 nuclease probing, and comparative sequence analysis identified a simple, 45 nt helical structure with several internal bulges as the core-binding element. These aptamers recognize with high specificity the isoalloxazine nucleus of FAD but do not distinguish FAD from FADH(2), nor are they removed from an FAD resin with UMP (which shares a pattern of hydrogen bond donors and acceptors along one face). Thus, these aptamers are structurally and functionally distinct from previously identified FMN and riboflavin aptamers. Circular dichroism data suggest a conformational change in the RNA upon FAD binding. These aptamers require magnesium and are active across a wide pH range (4.5-8.9). Since general acid-base catalysis plays a role in some flavin-dependent redox reaction mechanisms, these aptamers may be particularly well-suited to the design of new redox ribozymes.     

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.     

Coenzymes as coribozymes

Coenzymes are small organic molecules that supply a varied set of reactive groups to protein enzymes, thereby diversifying catalysis beyond the chemistries of amino acid sidechains. As RNA structures begin with a more limited chemical diversity than proteins, it seems likely that RNA enzymes would also use functional groups from other molecules to support a complex RNA world metabolism. In fact, ribonucleotide moieties in many coenzymes have long been thought to be surviving vestiges of covalently bound coenzymes in an RNA world. The idea of coenzyme utilization by ribozymes can be explored by selection-amplification of coenzyme-binding RNAs and coenzyme-assisted ribozymes. Here, we review coenzyme-RNAs, and discuss their possible significance for RNA-mediated metabolism. In summary, a plausible route from prebiotic chemistry to ribozyme biochemistry exists for CoA, and via similar activities, likely exists for all the nucleotidyl coenzymes.     

The role of metal ions in RNA catalysis

Understanding the catalytic mechanisms of RNA enzymes remains an important and intriguing challenge - one that has grown in importance since the recent demonstration that the ribosome is a ribozyme. At first, it seemed that all RNA enzymes compensate for the limited chemical versatility of ribonucleotide functional groups by recruiting obligatory metal ion cofactors to carry out catalytic chemistry. Mechanistic studies of the large self-splicing and pre-tRNA-processing ribozymes continue to support this idea, yielding increasingly detailed views of RNA active sites as scaffolds for positioning catalytic metal ions. Re-evaluation of the methodologies used to distinguish catalytic and structural roles for metal ions, however, has challenged this notion in the case of the small self-cleaving RNAs. Recent studies of the small ribozymes blur the distinction between catalytic and structural roles for metal ions, and suggest that RNA nucleobases have a previously unrecognized capacity for mediating catalytic chemistry.     

Ribozyme catalysis of metabolism in the RNA world

In vitro selection has proven to be a useful means of explore the molecules and catalysts that may have existed in a primordial 'RNA world'. By selecting binding species (aptamers) and catalysts (ribozymes) from random sequence pools, the relationship between biopolymer complexity and function can be better understood, and potential evolutionary transitions between functional molecules can be charted. In this review, we have focused on several critical events or transitions in the putative RNA world: RNA self-replication; the synthesis and utilization of nucleotide-based cofactors; acyl-transfer reactions leading to peptide and protein synthesis; and the basic metabolic pathways that are found in modern living systems.     

RNA-Catalyzed CoA, NAD, and FAD synthesis from phosphopantetheine, NMN, and FMN

A novel in vitro selection method was developed to isolate RNA sequences with coenzyme-synthesizing activities. We used size-heterogeneous libraries containing randomized ribonucleotide sequences of four different lengths (30N, 60N, 100N, and 140N), all with 5'-ATP initiation. Two RNAs, CoES7 (30N) and CoES21 (60N), are able to catalyze the synthesis of three common coenzymes, CoA, NAD, and FAD, from their precursors, 4'-phosphopantetheine, NMN, and FMN, respectively. Both ribozymes require divalent manganese for activities. The results support the availability of these coenzymes in an RNA world, and point to a chemical explanation for the complex bipartite structures of many coenzymes.     

Comparative study of DNA enzymes and ribozymes against the same full-length messenger RNA of the vanilloid receptor subtype I.

The efficiencies of 32 antisense oligodeoxynucleotides, 35 DNA enzymes and 6 ribozymes to bind and cleave the full-length messenger RNA of the vanilloid receptor subtype I were analyzed. Systematic screening of the mRNA revealed that good accessibility of a putative cleavage site for antisense oligodeoxynucleotides is a necessary but not a sufficient prerequisite for efficient DNA enzymes. Comparison of DNA enzymes and ribozymes against the same target sites revealed: 1) DNA enzymes were more active with longer recognition arms (9 nucleotides on either side), whereas ribozymes revealed higher activities with shorter recognition arms (7 nucleotides on either side). 2) It does not only depend on the target site but also on the enzyme sequence, whether a DNA enzyme or a ribozyme is more active. 3) The most efficient DNA enzyme found in this study had an approximately 15-fold higher reaction rate, k(react), and a 100-fold higher k(react)/K(m) under single turnover conditions compared with the fastest ribozyme. DNA enzymes as well as ribozymes showed significant activity under multiple turnover conditions, the DNA enzymes again being more active. We therefore conclude that DNA enzymes are an inexpensive, very stable and active alternative to ribozymes for the specific cleavage of long RNA molecules.     

In vitro selection of nucleoprotein enzymes.

Natural nucleic acids frequently rely on proteins for stabilization or catalytic activity. In contrast, nucleic acids selected in vitro can catalyze a wide range of reactions even in the absence of proteins. To augment selected nucleic acids with protein functionalities, we have developed a technique for the selection of protein-dependent ribozyme ligases. After randomizing a previously selected ribozyme ligase, L1, we selected variants that required one of two protein cofactors, a tyrosyl transfer RNA (tRNA) synthetase (Cyt18) or hen egg white lysozyme. The resulting nucleoprotein enzymes were activated several thousand fold by their cognate protein effectors, and could specifically recognize the structures of the native proteins. Protein-dependent ribozymes can potentially be adapted to novel assays for detecting target proteins, and the selection method's generality may allow the high-throughput identification of ribozymes capable of recognizing a sizable fraction of a proteome.     

Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA.

With the eventual goal of developing a treatment for chronic myelogenous leukemia (CML), attempts have been made to design hammerhead ribozymes that can specifically cleave BCR-ABL fusion mRNA. In the case of L6 BCR-ABL fusion mRNA (b2a2 type; BCR exon 2 is fused to ABL exon 2), which has no effective cleavage sites for conventional hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to cleave the chimeric mRNA specifically. Several hammerhead ribozymes with relatively long junction-recognition sequences have poor substrate-specificity. Therefore, we explored the possibility of using newly selected DNA enzymes that can cleave RNA molecules with high activity to cleave L6 BCR-ABL fusion (b2a2) mRNA. In contrast to the results with the conventional ribozymes, the newly designed DNA enzymes, having higher flexibility for selection of cleavage sites, were able to cleave this chimeric RNA molecule specifically at sites close to the junction. Cleavage occurred only within the abnormal BCR-ABL mRNA, without any cleavage of the normal ABL or BCR mRNA. Thus, these chemically synthesized DNA enzymes seem to be potentially useful for application in vivo , especially for the treatment of CML, if we can develop exogenous delivery strategies.     

Nuclease resistant ribozymes with high catalytic activity.

Hammerhead ribozymes are efficient RNA enzymes characterized by a typical hammerhead secondary structure and a number of conserved bases. Little is known about the role of the ribose-phosphate backbone, although it is obviously important since a DNA molecule with the same base sequence is not a catalyst. Here we describe the synthesis of artificial ribozymes where modified (2'-O-allyl- and 2'-O-methyl-) ribonucleotides substitute for the corresponding ribonucleotides. A systematic analysis of partially substituted polymers identified a minimum set of six non-contiguous positions where insertion of modified ribonucleotides strongly affects catalytic activity. Surprisingly, ribozymes completely substituted except for these six ribonucleotides are still very active. These molecules efficiently cleave in trans target RNAs in a sequence-specific way, but, unlike RNA ribozymes, are very resistant to nuclease degradation and are very stable in serum. These properties make such synthetic polymers potentially useful for in vivo gene expression studies and therapeutic applications.     

A general strategy for effector-mediated control of RNA-cleaving ribozymes and DNA enzymes

A novel and general approach is described for generating versions of RNA-cleaving ribozymes (RNA enzymes) and DNAzymes (DNA enzymes), whose catalytic activity can be controlled by the binding of activator molecules. Variants of the RNA-cleaving 10-23 DNAzyme and 8-17 DNAzyme were created, whose catalysis was activated by up to approximately 35-fold by the binding of the effector adenosine. The design of such variants was possible even though the tertiary folding of the two DNAzymes is not known. Variants of the hammerhead ribozyme were constructed, to respond to the effectors ATP and flavin mononucleotide. Whereas in conventional allosteric ribozymes, effector-binding modulates the chemical step of catalysis, here, effectors exercise their effect upon the substrate-binding step, by stabilizing the enzyme-substrate complex. Because such an approach for controlling the activity of DNAzymes/ribozymes requires no prior knowledge of the enzyme's secondary or tertiary folding, this regulatory strategy should be generally applicable to any RNA-cleaving ribozyme or DNAzyme, natural or in vitro selected, provided substrate-recognition is achieved by Watson-Crick base-pairing.     

Focus on function: single molecule RNA enzymology

The ability of RNA to catalyze chemical reactions was first demonstrated 25 years ago with the discovery that group I introns and RNase P function as RNA enzymes (ribozymes). Several additional ribozymes were subsequently identified, most notably the ribosome, followed by intense mechanistic studies. More recently, the introduction of single molecule tools has dissected the kinetic steps of several ribozymes in unprecedented detail and has revealed surprising heterogeneity not evident from ensemble approaches. Still, many fundamental questions of how RNA enzymes work at the molecular level remain unanswered. This review surveys the current status of our understanding of RNA catalysis at the single molecule level and discusses the existing challenges and opportunities in developing suitable assays.     

A common speed limit for RNA-cleaving ribozymes and deoxyribozymes

It is widely believed that the reason proteins dominate biological catalysis is because polypeptides have greater chemical complexity compared with nucleic acids, and thus should have greater enzymatic power. Consistent with this hypothesis is the fact that protein enzymes typically exhibit chemical rate enhancements that are far more substantial than those achieved by natural and engineered ribozymes. To investigate the true catalytic power of nucleic acids, we determined the kinetic characteristics of 14 classes of engineered ribozymes and deoxyribozymes that accelerate RNA cleavage by internal phosphoester transfer. Half approach a maximum rate constant of approximately 1 min(-1), whereas ribonuclease A catalyzes the same reaction approximately 80,000-fold faster. Additional biochemical analyses indicate that this commonly encountered ribozyme "speed limit" coincides with the theoretical maximum rate enhancement for an enzyme that uses only two specific catalytic strategies. These results indicate that ribozymes using additional catalytic strategies could be made that promote RNA cleavage with rate enhancements that equal those of proteins.