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At least 19 recordsLinked to original sources
PubMed1994

Three-dimensional working model of M1 RNA, the catalytic RNA subunit of ribonuclease P from Escherichia coli.

A three-dimensional model of M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, was constructed with the aid of a computer. The modeling process took into account data from chemical and enzymatic protection experiments, phylogenetic analysis, studies of the activities of mutants, and the kinetics of reactions catalyzed by the binding of substrate to M1 RNA. The model provides a plausible picture of the binding to M1 RNA of the tRNA domain of a precursor tRNA substrate. The scissile bond and adjacent segments of the aminoacyl acceptor stem of a precursor tRNA substrate can fit into a cleft that leads to the phylogenetically conserved, central part of the structure.

Base Sequence↗
PubMed1992

Important 2'-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli.

The role of 2'-hydroxyl groups in a model substrate for RNase P from Escherichia coli was studied using mixed DNA/RNA derivatives of such a substrate. The presence of the 2'-hydroxyl groups of nucleotides at positions -1 and -2 in the leader sequence and at position 1, as well as at the first C in the 3'-terminal CCA sequence, are important but not absolutely essential for efficient cleavage of the substrate by RNase P or its catalytic RNA subunit, M1 RNA. The 2'-hydroxyl groups in the substrate that are important for efficient cleavage also participate in the binding of Mg2+. An all-DNA external guide sequence (EGS) can efficiently render a potential substrate, derived from the model substrate, susceptible to cleavage by the enzyme or its catalytic RNA subunit. Furthermore, both DNA and RNA EGSs turn over during the reaction with RNase P in vitro. The identity of the nucleotide at position 1 in the substrate, the adjacent Mg(2+)-binding site in the leader sequence, and the junction of the single and double-stranded regions are the important elements in the recognition of model substrates, as well as in the identification of the sites of cleavage in those model substrates.

Base Sequence↗
PubMed1993

In vitro selection and evolution of RNA: applications for catalytic RNA, molecular recognition, and drug discovery.

In vitro selection and in vitro evolution methods represent powerful tools for isolating functional RNA molecules, and are proving to have wide applications in biology. Selection in the absence of living cells is possible because some RNA molecules possess a selectable "phenotype" (catalytic activity or ligand binding) as well as a "genotype" (nucleotide sequence). This review discusses the basic principles of in vitro selection technology and the application of these methods to isolate RNA molecules with interesting and novel properties. Selection techniques have been used to analyze the structure and function of catalytic RNA molecules (ribozymes), and to isolate novel catalytic structures not found in nature. They are also useful for studying protein-RNA interactions and for isolating RNA molecules that bind specifically to peptides and other ligands. The isolation of RNA molecules with new binding functionalities (aptamers) for both large and small molecules has exciting potential for discovery of new drugs and diagnostic reagents.

Base Sequence↗
PubMed2007

Potent knock down of HIV-1 replication by targeting HIV-1 Tat/Rev RNA sequences synergistically with catalytic RNA and DNA.

OBJECTIVE: Ribozymes (Rzs) and DNA-enzymes (Dzs) possess the ability to prevent gene expression by cleaving target RNA in a catalytic and sequence-specific manner. Although Rzs or Dzs have been used earlier for HIV-1 gene suppression, the present study explored the possibility of using catalytic RNA and DNA simultaneously in a synergistic manner with the hope that this novel approach will allow more potent inhibition for a longer duration. METHODS: In order to achieve long-term inhibition of HIV-1 replication, a novel non-GUX hammerhead Rz was designed by standard recombinant DNA technology and cloned it under the powerful CMV promoter containing expression vector. A 10-23 catalytic motif containing Dz that was targeted against the conserved second exon of HIV-1 Tat/Rev region was also assembled. RESULTS: Both Rz and Dz possessed sequence-specific cleavage activities individually and simultaneously cleaved target RNA in a synergistic manner under the same in vitro cleavage conditions. These catalytic molecules inhibited HIV-1 replication in macrophages individually and exhibited potent inhibitory effects when used in combination. CONCLUSIONS: The combination strategy described here can be widely used against any target RNA to achieve more effective gene inhibition that exploits the simultaneous sequence-specific cleavage potentials of catalytic RNA and DNA.

Amino Acid Sequence↗
PubMed2000

Recent insights on RNA folding mechanisms from catalytic RNA.

Methods for probing RNA structure in real time have revealed that initial folding steps are complete in less than a second. Refolding of large catalytic RNAs in vitro often results in long-lived intermediates that reach the native structure very slowly. These kinetically trapped intermediates arise from alternative secondary structures that form early in the folding process. In cells, proteins modulate the outcome of RNA folding reactions by stabilizing specific conformations or by accelerating refolding of misfolded intermediates. At the same time, competition between metastable conformations provides a means for regulating the biological activity of transcripts.

Animals↗
PubMed1996

Mapping in three dimensions of regions in a catalytic RNA protected from attack by an Fe(II)-EDTA reagent.

The accessibility of the ribose groups in the phosphodiester chain of M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli, has been probed with an Fe(II)-EDTA reagent when the RNA is alone in solution, when it is in a complex with a tRNA precursor substrate, and when it is in the holoenzyme complex with its cofactor, C5 protein. The regions found to be protected under these various conditions, as well as those previously identified in other chemical probing experiments, have been mapped on a three-dimensional working model of M1 RNA and are generally compatible with the previously proposed placement of the substrate on the enzyme and with previous data and inferences regarding the interactions of C5 protein with M1 RNA. On the basis of the accessibilities of the C(4') atoms, refinements have been introduced in the model to accommodate the Fe(II)-EDTA protection data. The protein cofactor makes contact with several helical regions of the catalytic RNA on the opposite side of the surface to which substrates bind.

Bacterial Proteins↗
PubMed1999

Verification of phylogenetic predictions in vivo and the importance of the tetraloop motif in a catalytic RNA.

M1 RNA, the catalytic subunit of Escherichia coli RNase P, forms a secondary structure that includes five sequence variants of the tetraloop motif. Site-directed mutagenesis of the five tetraloops of M1 RNA, and subsequent steady-state kinetic analysis in vitro, with different substrates in the presence and absence of the protein cofactor, reveal that (i) certain mutants exhibit defects that vary in a substrate-dependent manner, and that (ii) the protein cofactor can correct the mutant phenotypes in vitro, a phenomenon that is also substrate dependent. Thermal denaturation curves of tetraloop mutants that exhibit kinetic defects differ from those of wild-type M1 RNA. Although the data collected in vitro underscore the importance of the tetraloop motif to M1 RNA function and structure, three of the five tetraloops we examined in vivo are essential for the function of E. coli RNase P. The kinetic data in vitro are not in total agreement with previous phylogenetic predictions but the data in vivo are, as only mutants in those tetraloops proposed to be involved in tertiary interactions fail to complement in vivo. Therefore, the tetraloop motif is critical for the stabilization of the structure of M1 RNA and essential to RNase P function in the cell.

Base Sequence↗
PubMed1995

Processing of the precursor to the catalytic RNA subunit of RNase P from Escherichia coli.

M1 RNA, the catalytic subunit of RNase P from Escherichia coli, is transcribed in vivo as a precursor with extra nucleotides at the 3' end. Although it was suggested previously that RNase E is not responsible for the 3' processing of M1 RNA, we show that RNase E is the enzyme responsible for this reaction. At nonpermissive temperatures, the 3' processing of M1 RNA is abolished in a temperature-sensitive strain of E. coli that harbors a mutation in the gene for RNase E. Enhanced processing of M1 RNA is correlated with the overproduction of RNase E in vivo and processing is also correlated with the activity of this enzyme during the course of its purification. The biosynthesis of mature M1 RNA can proceed from transcripts that are produced under the control of a proximal promoter, as well as from a distal, upstream promoter. Transcription from the distal promoter results in a polycistronic transcript that includes four open reading frames and the transcript of rnpB, the gene coding for M1 RNA. The enzymatic activity that removes the 5' nucleotides from the precursor to M1 RNA is not due to RNase E, RNase P, or RNase III alone.

Base Sequence↗
PubMed1993

Product release is a rate-limiting step during cleavage by the catalytic RNA subunit of Escherichia coli RNase P.

The kinetic constants for cleavage of the tRNA(Tyr)Su3 precursor by the M1 RNA of E. coli RNase P were determined in the absence and presence of the C5 protein under single and multiple (steady state) turnover conditions. The rate constant of cleavage in the reaction catalyzed by M1 RNA alone was 5 times higher in single turnover than in multiple turnovers, suggesting that a rate-limiting step is product release. Cleavage by M1 RNA alone and by the holoenzyme under identical buffer conditions demonstrated that C5 facilitated product release. Addition of different product-like molecules under single turnover reaction conditions inhibited cleavage both in the absence and presence of C5. In the presence of C5, the Ki value for matured tRNA was approximately 20 times higher than in its absence, suggesting that C5 also reduces the interaction between the 5'-matured tRNA and the enzyme. In a growing cell the number of tRNA molecules is approximately 1000 times higher than the number of RNase P molecules. A 100-fold excess of matured tRNA over enzyme clearly inhibited cleavage in vitro. We discuss the possibility that RNase P is involved in the regulation of tRNA expression under certain growth conditions.

Base Sequence↗
PubMed1989

RNA catalytic properties of the minimum (-)sTRSV sequence.

We have identified an RNA catalytic domain within the sequence of the 359 base long negative-strand satellite RNA of tobacco ringspot virus. The catalytic domain contains two minimal sequences of satellite RNA, a 50-base catalytic RNA sequence, and a 14-base substrate RNA sequence. The catalytic complex of catalytic RNA/substrate RNA represents a structure not previously found in any RNA catalytic reaction described to date. The reaction is truly catalytic since the catalytic RNA has multiple substrate cleavage events and is not consumed during the course of the reaction. A linear relationship is seen between reaction rate and catalytic RNA concentration. The reaction has a Km of 0.03 microM, a kcat of 2.1/min, a temperature optimum of near 37 degrees C, and an energy of activation of 19 kcal/mol.

Base Sequence↗
PubMed2000

Effective inhibition of human cytomegalovirus gene expression and replication by a ribozyme derived from the catalytic RNA subunit of RNase P from Escherichia coli.

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping exon 3 region of the mRNAs encoding the major transcription regulatory proteins IE1 and IE2 of human cytomegalovirus. A reduction of more than 80% in the expression levels of IE1 and IE2 and a reduction of about 150-fold in viral growth were observed in human cells that stably expressed the ribozyme. In contrast, a reduction of less than 10% in the IE1/IE2 expression and viral growth was observed in cells that either did not express the ribozyme or produced a "disabled" ribozyme that carried mutations that abolished its catalytic activity. Examination of the expression of several other viral early and late genes in the cells that expressed the M1GS ribozyme further revealed an overall reduction of at least 80% in their expression. These results are consistent with the notion that the antiviral effects in these cells are due to the fact that the ribozyme specifically inhibits the expression of IE1 and IE2 and, consequently, abolishes the expression of viral early and late genes as well as viral growth. Our study is the first, to our knowledge, to use M1GS ribozyme for inhibiting human cytomegalovirus replication and demonstrates the utility of this ribozyme for antiviral applications.

Antiviral Agents↗
PubMed1990

Catalytic RNA and the origin of genetic systems.

The recent discovery of catalytic RNA molecules has provoked enormous interest in the origin of life and has given rise to new speculations about how living systems developed on the primitive earth. Here we outline why the discovery of catalytic RNA molecules has profound evolutionary implications, and then go on to discuss models for the emergence of metabolic complexity and protein synthesis in a primitive 'RNA world', emphasizing the constraints imposed on such models by genetical arguments.

Biological Evolution↗
PubMed1991

Visualizing the higher order folding of a catalytic RNA molecule.

The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry. The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions. Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed. Enzymatic activity, assayed with an RNA substrate that is complementary to the catalytic RNA active site, coincides with the cooperative structural transition. The higher order RNA foldings produced by Mg(II), Ca(II), and Sr(II) are similar; however, only the Mg(II)-stabilized RNA is catalytically active. Thus, these results directly demonstrate that divalent metal ions participate in general folding of the ribozyme tertiary structure, and further indicate a more specific involvement of Mg(II) in catalysis.

Animals↗