Substrate specificity and reaction kinetics of an X-motif ribozyme (original) (raw)
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Kinetic Analysis of δ Ribozyme Cleavage†
The ability of δ ribozyme to catalyze the cleavage of an 11-mer RNA substrate was examined under both single-and multiple-turnover conditions. In both cases only small differences in the kinetic parameters were observed in the presence of either magnesium or calcium as cofactor. Under multiple-turnover conditions, the catalytic efficiency of the ribozyme (k cat /K M) was higher at 37 °C than at 56 °C. The cleavage reaction seems to be limited by the product release step at 37 °C and by the chemical cleavage step at 56 °C. We observed substrate inhibition at high concentrations of the 11-mer substrate. Cleavage rate constants were determined with a structural derivative characterized by an ultrastable L4 tetraloop. The kinetic parameters (k cat and K M) and dissociation constant (K d) were almost identical for both ribozymes, suggesting that the stability of the L4 loop has a negligible impact on the catalytic activities of the examined ribozymes. Various cleavage inhibition and gel-shift assays with analogues, substrate, and both active and inactive ribozymes were performed. The 2′-hydroxyl group adjacent to the scissile phosphate was shown to be involved in binding with the ribozyme, while the essential cytosine residue of the J4/2 junction was shown to contribute to substrate association. We clearly show that substrate binding to the δ ribozyme is not restricted to the formation of a helix located downstream of the cleavage site. Using these results, we postulate a kinetic pathway involving a conformational transition step essential for the formation of the active ribozyme/substrate complex.
Ribozymes: recent advances in the development of RNA tools
FEMS Microbiology Reviews, 2003
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.
Tetrahedron, 1994
Small synthetic lariat RNAs have ken found to undergo site specific self-cleavage to give an acyclic branched-RNA with 2'3'~cyclic phosphate and a S-hydroxyl termird, which is reminiscent of tk products formed in some catalytic RNAs. These lariat-RNAs are much smaller than tk natural catalytic RNAs such as tk hammerkad ribosyme (k =-I mini at 37 7. and tkir rate of tk seIf-cleavage is also much slower (k = 0.25 x lo' mix' for larkt hexamer 18. and 0.16 x lO_ mitt-' for lariat kptamer 19 at 22 'C)). We have shown that tk trbmckotidyl loop in tk tetrameric and pentameric lariat-RNAs (ref. 10) is completely stable whereas the tetranucleotidyl or pentanucleotidyl loop in tk hexameric or kptameric lariat-RNA (ref. 10-13) does indeed have tk required local and global conformation promoting tk self-cleavage. It has ken also shown that simple 2'-tS or 3'+5'-lbtbd cyclic RNAs, I6 and 17. respectively, are completely stable and their structures are considerably di#erent from tk selfcleaving lariat-RNAs such as 18 or 19. In our search to ewlore tk optbnal structural requirementfor tk self-cleavage reaction of RNA, we have tww synthesized 14 in which tk branch-point adenosine has a 2'+5'-linkd tetranucleotidyl loop and a 3'-ethylphosphate moiety mimiching tk 3'-1ai1 of tk lariat-kxamer 18. We here report that tk unique 3'ethylphosphate function at the branch-point in 14 is the hey structural feature that orchestrates its self-cleavage reaction (k = 0.15 x 10" mini at 19 "c) compared to tk stable 2'+5'-linkd cyclic RNA 16 (see Fig. 1). We also report tk &tailed conformational features of tk self-cleaving tetrameric lariat-RNA 14 by 500 MHz NMR spectroscopy and Molecular dynamics simulations in tk aqueous environment. A comparative study of lhe temperature &pendence of tk N 2 S equilibrium for tk lariat-tetramer 14 and tk 2'-tS-l&d cyclic ietramer 16 shows that tk A' residue in 14 ls in 92% S-type conformation at 20 r. wkras it is only in 55% S in I6 with a 3'-hy&oxyl group. This displacement of tk N 2 S pseu&rotational equRibrium toward tk S geometry is due to tk enhanced gauche effect of tk 3'-OPO3Et group at tk branch-point adenosine in 14 compared to 3'-OH group in 16. This 3'-OPOsEtgroup promoted stabilization of tk S geometry at tk branch-point by AH = 4 kcal.mol-' in 14 is tk conformational driving force promoting its unique self-cleavage reaction. The comparison of AH' and AS" of the N 2 S pseudorotational equilibria in 14 and I6 (see Table 5) clearly shows tk remarkable effect of tk 3'-ethylphosphate group in 14 in being able to dictate tk ~~r&ormational changes from tk sugar moiety of tk branch-point adenosine to tk entire molecule (conformatio~l transmission). Thus tk S conformation in A', U2 and c6 sugar moieties is clearly tkrmodynamically more stabilized while it is considerably destabilized in d owing to tk 3'-ethylphosphate group in I4 compared to 16. It is interesting to note that tk magnitude of enthalpy and entropy for tk North to South transition of tk A1 sugar in 14 is comparable to the enthalpy and entropy of transition between tk A-and B-form of tk lariat hexamer 18 (ref. 12). This self-cleaving tetrameric lariat-RNA 14 is the smallest RNA molecule hitherto known to undergo tk self-cleavage reaction and hence it is the simplest model of tk active cleavage site of tk natural selfcleaving catalytic RNA. Most of the catalytically active natural RNA molecules are large and form complex tertiary structuresl-E. In several cases, the catalytic activity includes the site specific self-cleavage of a phosphodiester bond to give a 2',3'-cyclic phosphate and a S-hydroxyl terminus lv2. This self-cleavage reaction occurs by a transesterification
Ribozymes: the characteristics and properties of catalytic RNAs
FEMS Microbiology Reviews, 1999
Ribozymes, or catalytic RNAs, were discovered a little more than 15 years ago. They are found in the organelles of plants and lower eukaryotes, in amphibians, in prokaryotes, in bacteriophages, and in viroids and satellite viruses that infect plants. An example is also known of a ribozyme in hepatitis delta virus, a serious human pathogen. Additional ribozymes are bound to be found in the future, and it is tempting to regard the RNA component(s) of various ribonucleoprotein complexes as the catalytic engine, while the proteins serve as mere scaffolding^an unheard-of notion 15 years ago! In nature, ribozymes are involved in the processing of RNA precursors. However, all the characterized ribozymes have been converted, with some clever engineering, into RNA enzymes that can cleave or modify targeted RNAs (or even DNAs) without becoming altered themselves. While their success in vitro is unquestioned, ribozymes are increasingly used in vivo as valuable tools for studying and regulating gene expression. This review is intended as a brief introduction to the characteristics of the different identified ribozymes and their properties. ß Contents 0168-6445 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 -6 4 4 5 ( 9 9 ) 0 0 0 0 7 -8 * Tel.
Biochemistry, 1999
The hammerhead ribozyme crystal structure identified a specific metal ion binding site referred to as the P9/G10.1 site. Although this metal ion binding site is ∼20 Å away from the cleavage site, its disruption is highly deleterious for catalysis. Additional published results have suggested that the pro-R P oxygen at the cleavage site is coordinated by a metal ion in the reaction's transition state. Herein, we report a study on Cd 2+ rescue of the deleterious phosphorothioate substitution at the cleavage site. Under all conditions, the Cd 2+ concentration dependence can be accounted for by binding of a single rescuing metal ion. The affinity of the rescuing Cd 2+ is sensitive to perturbations at the P9/G10.1 site but not at the cleavage site or other sites in the conserved core. These observations led to a model in which a metal ion bound at the P9/G10.1 site in the ground state acquires an additional interaction with the cleavage site prior to and in the transition state. A titration experiment ruled out the possibility that a second tightbinding metal ion (K d Cd < 10 µM) is involved in the rescue, further supporting the single metal ion model. Additionally, weakening Cd 2+ binding at the P9/G10.1 site did not result in the biphasic binding curve predicted from other models involving two metal ions. The large stereospecific thio-effects at the P9/ G10.1 and the cleavage site suggest that there are interactions with these oxygen atoms in the normal reaction that are compromised by replacement of oxygen with sulfur. The simplest interpretation of the substantial rescue by Cd 2+ is that these atoms interact with a common metal ion in the normal reaction. Furthermore, base deletions and functional group modifications have similar energetic effects on the transition state in the Cd 2+ -rescued phosphorothioate reaction and the wild-type reaction, further supporting the model that a metal ion bridges the P9/G10.1 and the cleavage site in the normal reaction (i.e., with phosphate linkages rather than phosphorothioate linkages). These results suggest that the hammerhead undergoes a substantial conformational rearrangement to attain its catalytic conformation. Such rearrangements appear to be general features of small functional RNAs, presumably reflecting their structural limitations.
Modulating RNA structure and catalysis: lessons from small cleaving ribozymes
Cellular and Molecular Life Sciences, 2009
RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today's knowledge in the field.
The role of phosphate groups in the VS ribozyme-substrate interaction
Nucleic Acids Research, 2004
The VS ribozyme trans-cleavage substrate interacts with the catalytic RNA via tertiary interactions. To study the role of phosphate groups in the ribozyme-substrate interaction, 18 modified substrates were synthesized, where an epimeric phosphorothioate replaces one of the phosphate diester linkages. Sites in the stem-loop substrate where phosphorothioate substitution impaired reaction cluster in two regions. The first site is the scissile phosphate diester linkage and nucleotides downstream of this and the second site is within the loop region. The addition of manganese ions caused recovery of the rate of reaction for phosphorothioate substitutions between A621 and A622 and U631 and C632, suggesting that these two phosphate groups may serve as ligands for two metal ions. In contrast, significant manganese rescue was not observed for the scissile phosphate diester linkage implying that electrophilic catalysis by metal ions is unlikely to contribute to VS ribozyme catalysis. In addition, an increase in the reaction rate of the unmodified VS ribozyme was observed when a mixture of magnesium and manganese ions acted as the cofactor. One possible explanation for this effect is that the cleavage reaction of the VS ribozyme is rate limited by a metal dependent docking of the substrate on the ribozyme.
Biochemistry, 2006
As oxygen and selenium are in the same group (Family VI) in the Periodic Table, the site-specific mutagenesis at the atomic level by replacing RNA oxygen with selenium can provide insights on structure and function of catalytic RNAs. We report here the first Se-derivatized ribozymes transcribed with all nucleoside 5'-(α-P-seleno)triphosphates (NTPαSe, including A, C, G, and U). We found that T7 RNA polymerase recognizes NTP SeαSp diastereomers as well as the natural NTPs, while NTPαSe Rp diastereomers are neither substrates nor inhibitors. We also demonstrated the catalytic activity of these Se-derivatized hammerhead ribozymes by cleaving the RNA substrate, and we found that these phosphoroselenoate ribozymes can be as active as the native. These hammerhead ribozymes mutagenized site-specifically by selenium reveal the close relationship between the catalytic activities and the replaced oxygen atoms, which provides the insight of the oxygen participation in catalysis or intramolecular interaction. This demonstrates a convenient strategy for mechanistic study of functional RNAs. In addition, the active ribozymes derivatized sitespecifically by selenium will allow convenient MAD phasing in X-ray crystal structure study.
Nucleic Acids Research, 2002
A general approach is described for controlling the RNA-cleaving activity of nucleic acid enzymes (ribozymes and DNAzymes) via the use of oligonucleotide effectors (regulators). In contrast to the previously developed approaches of allosteric and facilitator-mediated regulation of such enzymes, this approach, called 'expansive' regulation, requires that the regulator bind simultaneously to both enzyme and substrate to form a branched three-way complex. Such three-way enzyme-substrate-regulator complexes are catalytically competent relative to the structurally unstable enzyme-substrate complexes. Using the 8-17 and bipartite DNAzymes and the hammerhead ribozyme as model systems, 20-to 30-fold rate enhancements were achieved in the presence of regulators of engineered variants of the above three enzymes, even under unoptimized conditions. Broadly, using this approach ribozyme and DNAzyme variants that are amenable to regulation by oligonucleotide effectors can be designed even in the absence of any knowledge of the folded structure of the relevant ribozyme or DNAzyme. Expansive regulation therefore represents a new and potentially useful technology for both the regulation of nucleic acid enzymes and the detection of specific RNA transcripts.
Nucleic Acids Research, 1996
The hairpin ribozyme is a small self-cleaving RNA that can be engineered for RNA cleavage in trans and has potential as a therapeutic agent. We have used a chemical synthesis approach to study the requirements of hairpin RNA cleavage for sugar and base moieties in residues of internal loop B, an essential region in one of the two ribozyme domains. Individual nucleosides were substituted by either a 2′-deoxynucleoside, an abasic residue, or a C3-spacer (propyl linker) and the abilities of the modified ribozymes to cleave an RNA substrate were studied in comparison with the wild-type ribozyme. From these results, together with previous studies, we propose a new model for the potential secondary structure of internal loop B of the hairpin ribozyme.