Self-Incorporation of coenzymes by ribozymes

RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes. Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities. The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function. In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNA-catalyzed incorporation of various coenzymes and coenzyme analogues. The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme. Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD⁺ and dephosphorylated CoA-SH. Similar ribozyme activities may have played an important role in the RNA world, when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.Correspondence to: G.F. Joyce     

RNA folding and catalysis

Catalysis in RNA is intimately connected to the folding. The small nucleolytic ribozymes function by a nucleophilic attack of the 2-oxygen on the 3-phosphate, in an S_N2 mechanism. This requires an alignment of the 2-O, 3-P and 5-O, that does not occur in normal A-form RNA. It is therefore likely that structural distortion plays a major role in the enhancement of the reaction rate, facilitating the trajectory into the in-line transition state. Given the polyelectrolyte nature of nucleic acids, metal ions are critical to folding processes in RNA. We have shown that two small nucleolytic ribozymes, the hammerhead and hairpin ribozymes, undergo metal ion-induced folding processes. The hammerhead ribozyme folds in two stages, each of which is induced by the binding of a single structural ion. The first corresponds to the formation of the ribozyme scaffold, while the second is the formation of the catalytic core of the ribozyme. By contrast, the hairpin ribozyme undergoes a single folding event induced by the binding of at least two metal ions, and involves the close interaction between two internal loops to form the active ribozyme.This revised version was published online in October 2005 with corrections to the Cover Date.     

A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.     

Feedback regulation of RNA polymerase subunit synthesis after the conditional overproduction of RNA polymerase in Escherichia coli

Summary The and subunit of RNA polymerase are thought to be controlled by a translational feedback mechanism regulated by the concentration of RNA polymerase holoenzyme. To study this regulation in vivo, an inducible RNA polymerase overproduction system was developed. This system utilizes plasmids from two incompatibility groups that carry RNA polymerase subunit genes under lac promoter/operator control. When the structural genes encoding the components of core RNA polymerase (, and ) or holoenzyme (, , and ⁷⁰) are present on the plasmids, induction of the lac promoter results in a two fold increase in the concentration of functional RNA polymerase. The induction of RNA polymerase overproduction is characterized by an initial large burst of synthesis followed by a gradual decrease as the concentration of RNA polymerase increases. Overproduction of RNA polymerase in a strain carrying an electrophoretic mobility mutation in the rpoB gene results in the specific repression of synthesis off the chromosome. These results indicate that RNA polymerase feedback regulation controls synthesis in vivo.     

RNA double cleavage by a hairpin-derived twin ribozyme

The hairpin ribozyme is a small catalytic RNA that catalyses reversible sequence-specific RNA hydrolysis in trans. It consists of two domains, which interact with each other by docking in an antiparallel fashion. There is a region between the two domains acting as a flexible hinge for interdomain interactions to occur. Hairpin ribozymes with reverse-joined domains have been constructed by dissecting the domains at the hinge and rejoining them in reverse order. We have used both the conventional and reverse-joined hairpin ribozymes for the design of a hairpin-derived twin ribozyme. We show that this twin ribozyme cleaves a suitable RNA substrate at two specific sites while maintaining the target specificity of the individual monoribozymes. For characterisation of the studied ribozymes we have evaluated a quantitative assay of sequence-specific ribozyme activity using fluorescently labelled RNA substrates in conjunction with an automated DNA sequencer. This assay was found to be applicable with hairpin and hairpin-derived ribozymes. The results demonstrate the potential of hairpin ribozymes for multi-target strategies of RNA cleavage and suggest the possibility for employing hairpin-derived twin ribozymes as powerful tools for RNA manipulation in vitro and in vivo.     

Twin ribozyme mediated removal of nucleotides from an internal RNA site

Over the past two decades, the structure and mechanism of catalytic RNA have been extensively studied; now ribozymes are understood well enough to turn them into useful tools. After we have demonstrated the twin ribozyme mediated insertion of additional nucleotides into a predefined position of a suitable substrate RNA, we here show that a similar type of twin ribozyme is also capable of mediating the opposite reaction: the site-specific removal of nucleotides. In particular, we have designed a twin ribozyme that supports the deletion of four uridine residues from a given RNA substrate. This reaction is a kind of RNA recombination that in the specific context of gene therapy mimics, at the level of RNA, the correction of insertion mutations. As a result of the twin ribozyme driven reaction, 17% of substrate are converted into the four nucleotides shorter product RNA.     

The search for missing links between self-replicating nucleic ACIDs and the RNA world

The notion that modern metabolism was derived from a complex RNA world in which most reactions were catalyzed by ribozymes has received wide acceptance. However, the evolutionary links between the first self-replicating systems and ribozymes as complex as, say, the Group I self-splicing intron or the HDV ribozyme, have remained elusive. While prebiotic chemists have succeeded in synthesizing short oligonucleotides, it is not immediately obvious how these could have replicated and evolved to the point where they could assume complex shapes and catalytic functions. Nonetheless, recent experiments from a variety of disciplines suggest a plausible pathway from prebiotic chemistry to complex metabolism, and this review is intended as a hypothetical roadmap for the origin and subsequent evolution of life.     

Efficient RNA ligation by reverse-joined hairpin ribozymes and engineering of twin ribozymes consisting of conventional and reverse-joined hairpin ribozyme units

In recent years major progress has been made in elucidating the mechanism and structure of catalytic RNA molecules, and we are now beginning to understand ribozymes well enough to turn them into useful tools. Work in our laboratory has focused on the development of twin ribozymes for site-specific RNA sequence alteration. To this end, we followed a strategy that relies on the combination of two ribozyme units into one molecule (hence dubbed twin ribozyme). Here, we present reverse-joined hairpin ribozymes that are structurally optimized and which, in addition to cleavage, catalyse efficient RNA ligation. The most efficient variant ligated its appropriate RNA substrate with a single turnover rate constant of 1.1 min(-1) and a final yield of 70%. We combined a reverse-joined hairpin ribozyme with a conventional hairpin ribozyme to create a twin ribozyme that mediates the insertion of four additional nucleotides into a predetermined position of a substrate RNA, and thus mimics, at the RNA level, the repair of a short deletion mutation; 17% of the initial substrate was converted to the insertion product.     

Phytochrome-mediated regulation of β-amylase mRNA level in mustard (Sinapis alba L.) cotyledons

Phytochrome, activated by continuous red light, increases the amount of total polyadenylated RNA during photomorphogenesis of mustard (Sinapis alba L.) cotyledons. In-vitro translation of total polyadenylated RNA in a reticulocyte translation system has shown that the activity of translatable -amylase mRNA is increased by phytochrome about threefold in the 3-d-old cotyledons, based on equal amounts of polyadenylated RNA, and about eightfold on a per-cotyledon basis. Cordycepin prevents the accumulation of translatable -amylase mRNA. It is concluded that the phytochrome-mediated control of -amylase synthesis is exerted on the level of mRNA synthesis. During seedling development in continuous red light, a phytochrome-dependent increase of -amylase mRNA can be observed at least 6 h before the onset of -amylase synthesis. If, after a period of enzyme synthesis, phytochrome action is interrupted by long-wavelength far-red light followed by darkness, -amylase mRNA as well as -amylase synthesis remain at a high level for 8–10 h and then decline sharply. It is concluded that -amylase mRNA, having an apparent lifetime of the order of 8–10 h, can be formed under the influence of phytochrome during early seedling development but it activates -amylase synthesis only after a lag-phase of about 8 h, when the cotyledons acquire competence to synthesize the enzyme. The consequences of these findings for the signal-transduction chain of phytochrome are discussed.Abbreviations EDTA Na₂-ethylenediaminotetraacetic acid - PAGE polyacrylamide gel electrophoresis - poly(A)⁺RNA polyadenylated mRNA - P_r, P_fr red- and far-red-absorbing forms of phytochrome - SDS sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.     

Pattern generation in molecular evolution: Exploitation of the variation in RNA landscapes

Evolution of RNA secondary structure is studied using simulation techniques and statistical analysis of fitness landscapes. The transition from RNA sequence to RNA secondary structure leads to fitness landscapes that have local variations in their ruggedness. Evolution exploits these variations. In stable environments it moves the quasispecies toward relatively flat peaks, where not only the master sequence but also its mutants have a high fitness. In a rapidly changing environment, the situation is reversed; evolution moves the quasispecies to a region where the correlation between secondary structures of neighboring RNA sequences is relatively low. In selection for simple secondary structures the movement toward flat peaks leads to pattern generation in the RNA sequences. Patterns are generated at the level of polynucleotide frequencies and the distribution of purines and pyrimidines. The patterns increase the modularity of the sequence. They thereby prevent the formation of alternative secondary structures after mutations. The movement of the quasispecies toward relatively rugged parts of the landscape results in pattern generation at the level of the RNA secondary structure. The base-pairing frequency of the sequences increases. The patterns that are generated in the RNA sequences and the RNA secondary structures are not directly selected for and can be regarded as a side effect of the evolutionary dynamics of the system. Correspondence to: M.A. Huynen     

Brief fasting decreases protein synthesis in the brain of adult rats

The influence of starvation on protein synthesis in the adult rat brain was studied in vivo by an intravenous injection of a flooding dose of unlabeled valine including a tracer dose ofL-[3,4(n)-³H]valine. Brief starvation (24 hours) induced a 20% decline in fractional and absolute rates of brain protein synthesis. This decline resulted from a 20% decrease in the efficiency of protein synthesis (g protein synthesized per day per g RNA) whereas the capacity for protein synthesis (g RNA per mg protein) was maintained. Prolonged starvation (5 days) was marked by no further significant changes in the fractional rate, absolute rate and efficiency of protein synthesis, whereas the capacity for protein synthesis cecreased slightly. The relative contribution of brain to wholebody body protein synthesis increased during fasting, and neither the protein nor the RNA brain content did change during the experiment. These results clearly indicate that brain proteins are spared in response to brief and prolonged food deprivation, and that brain protein synthesis is very sensitive to short-term fasting.     

The many faces of the hairpin ribozyme: structural and functional variants of a small catalytic RNA

The hairpin ribozyme is a small catalytic RNA that has been reengineered resulting in a number of variants with extended or even new functions. Thus, manipulation of the hairpin ribozyme structure has allowed for activity control by external effectors, namely oligonucleotides, flavine mononucleotide, and adenine. Hairpin ribozyme-derived twin ribozymes that mediate RNA fragment exchange reactions as well as self-processing hairpin ribozymes were designed. Furthermore, several hairpin ribozyme variants have been engineered for knock down of specific RNA substrates by adapting the substrate-binding domain to the specific target sequence. This review will focus on hairpin ribozymes possessing structural extensions/variations and thus functionally differing from the parent hairpin ribozyme.     

Catalytic sites ofEscherichia coli F1-ATPase

The catalytic site ofEscherichia coli F₁-ATPase is reviewed in terms of structure and function. Structural prediction, biochemical analyses, and mutagenesis experiments suggest that the catalytic site is formed primarily by residues 137–335 of -subunit. Subdomains of the site involved in phosphate-bond cleavage/synthesis and adenine-ring binding are discussed. Ambiguities inherent in steady-state catalytic measurements due to catalytic site cooperativity are discussed, and the advantages of pre-steady-state (unisite) techniques are emphasized. The emergence of a single high-affinity catalytic site occurs as a result of F₁-oligomer assembly. Measurements of unisite catalysis rate and equilibrium constants, and their modulation by varied pH, dimethylsulfoxide, and mutations, are described and conclusions regarding the nature of the high-affinity catalytic site and mechanism of catalysis are presented.     

Chromatographic separation of DNA dependent RNA polymerases and molecular properties of RNA polymerase II from a Leishmania Spp

Multiple forms of DNA-dependent RNA polymerases have been isolated and characterized from Leishmania strain UR6 promastigotes. RNA polymerases from this organism fail to resolve into multiple forms by conventional chromatography on DEAE-Sephadex A25, but could be separated by a modification of the method using CM-Sephadex C25. The CM-Sephadex bound enzyme is resistant toamanitin even up to a concentration of 250g/ml. The activity which flows through CM-Sephadex further resolves into two forms upon chromatography on DEAE-Sephadex A25. These forms are sensitive to -amanitin to different extent. Enzyme activity in peak I is 50% inhibited by 3g/ml and in peak II by 50g/ml of the drug respectively. The enzyme in peak I has been further purified by heparin agarose and fast performance liquid chromatography (FPLC) on MonoQ. The enzyme has Stoke's radius of 70å, a sedimentation coefficient of 17.6S and an f/fo of 1.35. Analysis of ammonium sulfate and met n peak I, relative activities with Mn+2 versus Mg+2 and template specificities gave results similar to those reported for other type II RNA polymerases in eukaryotes. The MonoQ purified enzyme resolves into 16 polypeptides on denaturing polyacrylamide gel and densitometric analysis suggests that 9 major bands are present in the stoichiometry expected of RNA polymerase subunits having molecular weights: 154000; 104000; 77000; 64000; 52000; 48000; 46000; 45000 and 39000 respectively.     

Biochemical analysis of pistol self-cleaving ribozymes

Pistol RNAs are members of a distinct class of self-cleaving ribozymes that was recently discovered by using a bioinformatics search strategy. Several hundred pistol ribozymes share a consensus sequence including 10 highly conserved nucleotides and many other modestly conserved nucleotides associated with specific secondary structure features, including three base-paired stems and a pseudoknot. A representative pistol ribozyme from the bacterium Lysinibacillus sphaericus was found to promote RNA strand scission with a rate constant of ∼10 min⁻¹ under physiological Mg²⁺ and pH conditions. The reaction proceeds via the nucleophilic attack of a 2′-oxygen atom on the adjacent phosphorus center, and thus adheres to the same general catalytic mechanism of internal phosphoester transfer as found with all other classes of natural self-cleaving ribozymes discovered to date. Analyses of the kinetic characteristics and the metal ion requirements of the cleavage reaction reveal that members of this ribozyme class likely use several catalytic strategies to promote the rapid cleavage of RNA.     

Ribozymes: recent advances in the development of RNA tools

The discovery 20 years ago that some RNA molecules, called ribozymes, are able to catalyze chemical reactions was a breakthrough in biology. Over the last two decades numerous natural RNA motifs endowed with catalytic activity have been described. They all fit within a few well-defined types that respond to a specific RNA structure. The prototype catalytic domain of each one has been engineered to generate trans-acting ribozymes that catalyze the site-specific cleavage of other RNA molecules. On the 20th anniversary of ribozyme discovery we briefly summarize the main features of the different natural catalytic RNAs. We also describe progress towards developing strategies to ensure an efficient ribozyme-based technology, dedicating special attention to the ones aimed to achieve a new generation of therapeutic agents.     

Long-range impact of peripheral joining elements on structure and function of the hepatitis delta virus ribozyme

The HDV ribozyme is an RNA enzyme from the human pathogenic hepatitis delta virus (HDV) that has recently also been identified in the human genome. It folds into a compact, nested double-pseudoknot. We examined here the functional relevance of the capping loop L4 and the helical crossover J1/2, which tightly interlace the two helical stacks of the ribozyme. Peripheral structural elements such as these are present in cis-acting, but not trans-acting ribozymes, which may explain the order-of-magnitude decrease in cleavage activity observed in trans-acting ribozymes with promise in gene therapy applications. Comparison of a systematic set of cis- and trans-acting HDV ribozymes shows that the absence of either L4 or J1/2 significantly and independently impacts catalytic activity. Using terbium(III) footprinting and affinity studies, as well as distance measurements based on time-resolved fluorescence resonance energy transfer, we find that J1/2 is most important for conferring structural properties similar to those of the cis-acting ribozyme. Our results are consistent with a model in which removal of either a helical crossover or surprisingly a capping loop induces greater dynamics and expansion of the catalytic core at long range, impacting local and global folding, as well as catalytic function.     

Enzymatic cleavage of RNA by RNA

The discovery and characterization of the catalytic RNA subunit of the enzyme ribonuclease P ofEscherichia coli is described.Nobel lecture given on December 8, 1989, by Professor Sidney Altman, and published in LES PRIX NOBEL 1989, printed in Sweden by Norstedts Tryckeri, Stockholm, Sweden, 1990, republished here with the permission of the Nobel Foundation, the copyright holder.