Bidirectional effectors of a group I intron ribozyme (original) (raw)
Related papers
Implication of Arginyl Residues in mRNA Binding to Ribosomes
European Journal of Biochemistry, 1980
Modification of Esclzevichia coli ribosomes with phenylglyoxal and butanedione, protein reagents specific for arginyl residues, inactivates polypeptide polymerization, assayed as poly(U)dependent polyphenylalanine synthesis, and the binding of poly(U). Inactivation is produced by modification of the 30-S subunit. Both the RNA and the protein moieties of 30-S subunits are modified by phenylglyoxal, and modification of either of them is accompanied by inactivation of polypeptide synthesis. Modification of only the split proteins released from 30-S subunits by prolonged dialysis against a low-ionic-strength buffer, which contain mainly protein S1, produces inhibition of poly(U) binding and inactivation of polypeptide synthesis. Amino acid analysis of the modified split proteins showed a significant modifications of arginyl residues. These results indicate that the arginyl residues of a few 30-S proteins might be important in the interaction between mRNA and the 30-S subunit, which agrees with the general role assigned to the arginyl residues of proteins as the positively charged recognition site for anionic ligands.
Arginine Cofactors on the Polymerase Ribozyme
PLoS ONE, 2011
The RNA world hypothesis states that the early evolution of life went through a stage in which RNA served both as genome and as catalyst. The central catalyst in an RNA world organism would have been a ribozyme that catalyzed RNA polymerization to facilitate self-replication. An RNA polymerase ribozyme was developed previously in the lab but it is not efficient enough for self-replication. The factor that limits its polymerization efficiency is its weak sequence-independent binding of the primer/template substrate. Here we tested whether RNA polymerization could be improved by a cationic arginine cofactor, to improve the interaction with the substrate. In an RNA world, amino acid-nucleic acid conjugates could have facilitated the emergence of the translation apparatus and the transition to an RNP world. We chose the amino acid arginine for our study because this is the amino acid most adept to interact with RNA. An arginine cofactor was positioned at ten different sites on the ribozyme, using conjugates of arginine with short DNA or RNA oligonucleotides. However, polymerization efficiency was not increased in any of the ten positions. In five of the ten positions the arginine reduced or modulated polymerization efficiency, which gives insight into the substrate-binding site on the ribozyme. These results suggest that the existing polymerase ribozyme is not well suited to using an arginine cofactor.
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.
Ribozymes and Riboswitches: Modulation of RNA Function by Small Molecules
Biochemistry, 2010
Diverse small molecules interact with catalytic RNAs (ribozymes) as substrates and cofactors, and their intracellular concentrations are sensed by gene-regulatory mRNA domains (riboswitches) that modulate transcription, splicing, translation, or RNA stability. Although recognition mechanisms vary from RNA to RNA, structural analyses reveal recurring strategies that arise from the intrinsic properties of RNA such as base pairing and stacking with conjugated heterocycles, and cation-dependent recognition of anionic functional groups. These studies also suggest that, to a first approximation, the magnitude of ligand-induced reorganization of an RNA is inversely proportional to the complexity of the riboswitch or ribozyme. How these small molecule bindinginduced changes in RNA lead to alteration in gene expression is less well understood. While different riboswitches have been proposed to be under either kinetic or thermodynamic control, the biochemical and structural mechanisms that give rise to regulatory consequences downstream of small molecule recognition by RNAs mostly remain to be elucidated. Ribozymes and riboswitches starkly demonstrate the ability of RNA to fold into complex structures that position functional groups with exquisite precision. The former are catalytic RNAs; the latter, cis-acting regulatory mRNA domains that respond to the intracellular concentration of small molecule metabolites and second messengers [the first example of a transacting riboswitch RNA was recently described (1)]. In vitro, both ribozymes and riboswitches can function in the absence of protein cofactors, although some catalytic RNAs are known to require chaperones [reviewed in (2)] for in vivo activity, and riboswitches ultimately need to interface with the rest of the gene expression (transcription, splicing, translation, or RNA degradation) machinery for their small molecule-dependent regulatory activity to become manifest. Over the past decade, structural analyses have shed light on the mechanism of small molecule recognition by ribozymes (as substrates and coenzymes) and riboswitches (as regulatory signals). We review the state of knowledge of small molecule recognition by RNA, and how small molecule binding gives rise to genetic regulation.
Biochemistry, 1997
The recent increase in the population of immunocompromised patients has led to an insurgence of opportunistic human fungal infections. The lack of effective treatments against some of these pathogens makes it important to develop new therapeutic strategies. One such strategy is to target key RNAs with antisense compounds. We report the development of a model system for studying the potential for antisense targeting of group I self-splicing introns in fungal pathogens. The group I intron from the large ribosomal subunit RNA of mouse-derived Pneumocystis carinii has been isolated and characterized. This intron self-splices in Vitro. A catalytically active ribozyme, P-8/4x, has been constructed from this intron to allow measurement of dissociation constants for potential antisense agents. At 37°C, in 50 mM Hepes (25 mM Na + ), 15 mM MgCl 2 , and 135 mM KCl at pH 7.5, the exogenous 5′ exon mimic r(AUGACU) binds about 60 000 times more tightly to this ribozyme than to r(GGUCAU), a mimic of its complementary binding site on the ribozyme. This enhanced binding is due to tertiary interactions. This tertiary stabilization is increased by single deoxynucleotide substitutions in the exon mimic at every position except for the internal A, which is essentially unchanged. Thus 2′ OH groups of the 5′ exon mimic do not form stabilizing tertiary interactions with the P-8/4x ribozyme, in contrast to the Tetrahymena L-21 ScaI ribozyme. Furthermore, at 37°C, the exogenous 5′ exon mimic d(ATGACT) binds nearly 32 000 times more tightly to the P-8/4x ribozyme than to r(GGUCAU). Therefore, oligonucleotides without 2′ OH groups can exploit tertiary stabilization to bind dramatically more tightly and with more specificity than possible from base pairing. These results suggest a new paradigm for antisense targeting: targeting the tertiary interactions of structural RNAs with short antisense oligonucleotides.
Chemical models for ribozyme action
Current Opinion in Chemical Biology, 2005
Mechanistic studies of the action of catalytic ribonucleic acids, ribozymes, are highly challenging, because even a slight structural change can dramatically affect the chain folding. This, in turn, alters the binding properties of the catalytic core, making identification of the real origin of the observed influence on rate difficult. Unambiguous structure-reactivity correlations based on studies with structurally simplified chemical models may help to distinguish between alternative mechanistic interpretations. The results of such model studies are reviewed. The topics include intramolecular cleavage of RNA phosphodiester bonds by solvent-derived species, general acids/bases and metal ions, effect of molecular environment on their hydrolytic stability and trinucleoside monophosphates as models for large ribozymes.
Ribozymes: biology, biochemistry, and implications for clinical medicine
Journal of molecular …, 1995
Ribozymes are a class of ribonucleic acid (RNA) molecules that possess enzymatic properties. Upon binding to complementary nucleic acid strands, catalytic degradation takes place via a cleavage reaction. In effect, inactivation of susceptible substrate RNA molecules takes place at a catalytic rate and with a high degree of substrate specificity. This article reviews the biology and biochemistry of this class of molecules and its potential applications in clinical medicine.
Biochemistry, 2008
In the trans excision-splicing reaction, a Pneumocystis carinii group I intron-derived ribozyme binds an RNA substrate, excises a specific internal segment, and ligates the flanking regions back together. This reaction can occur both in vitro and in vivo. In this report, the first of the two reaction steps was analyzed to distinguish between two reaction mechanisms: ribozyme-mediated hydrolysis and nucleotidedependent intramolecular transesterification. We found that the 3′-terminal nucleotide of the ribozyme is the first-reaction step nucleophile. In addition, the 3′-half of the RNA substrate becomes covalently attached to the 3′-terminal nucleotide of the ribozyme during the reaction, both in vitro and in vivo. Results also show that the identity of the 3′-terminal nucleotide influences the rate of the intramolecular transesterification reaction, with guanosine being more effective than adenosine. Finally, expected products of the hydrolysis mechanism do not form during the reaction. These results are consistent with only the intramolecular transesterification mechanism. Unexpectedly, we also found that ribozyme constructs become truncated in vivo, probably through intramolecular 3′-hydrolysis (self-activation), to create functional 3′-terminal nucleotides.
Specificity of arginine binding by the Tetrahymena intron
Biochemistry, 1989
L-Arginine competitively inhibits the reaction of GTP with the Tetrahymena ribosomal self-splicing intron. In order to define this R N A binding site for arginine, Ki's have now been measured for numerous arginine-like competitive inhibitors. Detailed consideration of the Ki's suggests a tripartite binding model. L-and D-arginine from Chemical Dynamics, Fluka, and Sigma were compared without detectable differences. Guanidinoformic acid, guanidinoacetic acid, methylguanidine hydrochloride, ethylguanidine hydrochloride, butylguanidine hydrochloride, N"-acetyl-L-arginine, N"-benzoyl-L-arginyl ethyl ester, 6-guanidinocaproic acid, and 5-guanidinovaleric acid were obtained from Aldrich or the Bader Library of Rare Chemicals. L-2-Amino-3-guanidinopropionic acid and L-2amino-4-guanidinobutyric acid were from Chemical Dynamics. Guanylurea sulfate was obtained from Eastman Kodak. Agmatine, L-argininamide, L-arginine methyl ester, L-arginine ethyl ester, L-
Binding and cleavage of nucleic acids by the "hairpin" ribozyme
Biochemistry, 1991
The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Tritz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A,) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at AI is cleaved; thus AI provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A, on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of AI functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-R, oxygen at the scissile phosphodiester does not coordinate Mg2+. Ribozymes (RNA enzymes) catalyze site-specific RNA cleavage and ligation reactions. In contrast to most protein I Abbreviations: (-)sTRSV, negative RNA strand of the satellite RNA of tobacco ringspot virus.