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C Guerrier-Takada

Publications and source records attributed to C Guerrier-Takada.

At least 19 recordsLinked to original sources
PubMed1995

Artificial regulation of gene expression in Escherichia coli by RNase P.

Plasmids encoding various external guide sequences (EGSs) were constructed and inserted into Escherichia coli. In strains harboring the appropriate plasmids, the expression of fully induced beta-galactosidase and alkaline phosphatase activity was reduced by more than 50%, while no reduction in such activity was observed in strains with non-specific EGSs. The inhibition of gene expression was virtually abolished at restrictive temperatures in strains that were temperature-sensitive for RNase P (EC 3.1.26.5). Northern blot analysis showed that the steady-state copy number of EGS RNAs was several hundred per cell in vivo. A plasmid that contained a gene for M1 RNA covalently linked to a specific EGS reduced the level of expression of a suppressor tRNA that was encoded by a separate plasmid. Similar methods can be used to regulate gene expression in E. coli and to mimic the properties of cold-sensitive mutants.

Base Sequence↗
PubMed1992

Targeted cleavage of mRNA in vitro by RNase P from Escherichia coli.

External guide sequences (EGSs) complementary to mRNAs that encode beta-galactosidase from Escherichia coli and nuclease A from Staphylococcus aureus can target these RNAs for cleavage in vitro by RNase P from E. coli. Specific cleavage occurs at locations predicted by the nucleotide sequences of the EGSs. EGSs with regions complementary to the mRNAs that are as short as 13 nucleotides function efficiently and turn over slowly during incubation with the target substrate and the enzyme. EGSs composed of deoxyribonucleotides as well as those composed of ribonucleotides are effective, but cleavage of the targeted substrate with DNA as an EGS is about 10-fold less efficient than that with RNA as an EGS. An RNA EGS inhibited the formation of beta-galactosidase activity in a crude extract (S30) of E. coli that was capable of catalyzing coupled transcription-translation reactions.

Base Sequence↗
PubMed1992

Reconstitution of enzymatic activity from fragments of M1 RNA.

Certain fragments of M1 RNA, the catalytic subunit of RNase P from Escherichia coli, either have no enzymatic activity at all or have altered substrate specificity compared with that of the intact catalytic RNA. After simple mixing in vitro, many of these fragments of M1 RNA can reassociate with other fragments to form complexes that have enzymatic activity typical of wild-type M1 RNA. Furthermore, inactive M1 RNA molecules with internal deletions can be complemented in vitro by other inactive derivatives of M1 RNA that have nonoverlapping deletions. Thus, two inactive molecules of M1 RNA can interact to form an active RNA enzyme. Functional attributes can be assigned to various regions of M1 RNA when the reconstitution process is combined with assays for activity with different substrates.

Base Sequence↗
PubMed1990

Interaction of RNase P from Escherichia coli with pseudoknotted structures in viral RNAs.

In a previous study it was shown that RNase P from E. coli cleaves the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA in vitro (Guerrier-Takada et al. (1988) Cell, 53, 267-272). Cleavage takes place at the 3' side of the loop that crosses the deep groove of the pseudoknot structure present in the aminoacyl acceptor domain. In the present study fragments of TYMV RNA with mutations in the pseudoknot, generated by transcription in vitro, were tested for susceptibility to cleavage by RNase P. Changes in the specificity with respect to the site of cleavage and decreases in the rate of cleavage were observed with most of these substrates. The behaviour of various mutants in the reaction catalyzed by RNase P is in agreement with the present model of the TYMV RNA pseudoknot (Dumas et al. (1987), J. Biomol. Struct. Dyn. 263, 652-657). Base substitutions in the loop that crosses the shallow groove of the pseudoknot structure resulted, however, in an unexpected decrease in the rate of cleavage, probably due to conformational changes in the substrates. Studies on other tRNA-like structures revealed an important role in the reaction with RNase P for both the nucleotide at the 3' side of the loop that spans the deep groove and the nucleotide at position 4, which correspond to positions--1 and 73, respectively, in tRNA precursors.

Base Sequence↗
PubMed1989

Specific interactions in RNA enzyme-substrate complexes.

Analysis of crosslinked complexes of M1 RNA, the catalytic RNA subunit of ribonuclease P from Escherichia coli, and transfer RNA precursor substrates has led to the identification of regions in the enzyme and in the substrate that are in close physical proximity to each other. The nucleotide in M1 RNA, residue C92, which participates in a crosslink with the substrate was deleted and the resulting mutant M1 RNA was shown to cleave substrates lacking the 3' terminal CCAUCA sequence at sites several nucleotides away from the normal site of cleavage. The presence or absence of the 3' terminal CCAUCA sequence in transfer RNA precursor substrates markedly affects the way in which these substrates interact with the catalytic RNA in the enzyme-substrate complex. The contacts between wild-type M1 RNA and its substrate are in a region that resembles part of the transfer RNA "E" (exit) site in 23S ribosomal RNA. These data demonstrate that in RNA's with very different cellular functions, there are domains with similar structural and functional properties and that there is a nucleotide in M1 RNA that affects the site of cleavage by the enzyme.

Base Sequence↗
PubMed1989

Catalysis by the RNA subunit of RNase P--a minireview.

RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5' termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate.

Catalysis↗
PubMed1988

Novel reactions of RNAase P with a tRNA-like structure in turnip yellow mosaic virus RNA.

A quasi-continuous double hellix, containing a pseudoknot and ending in a single-stranded region which contains CCA, can be formed at the 3' terminus of the genomic RNAs of certain plant viruses. M1 RNA (the catalytic subunit) alone and the RNAase P holoenzyme from E. coli cleave the tRNA-like structure of TYMV RNA in vitro at the 5' side of the quasi-helical structure to generate 5' phosphate and 3' hydroxyl groups in the cleavage products. The intact pseudoknot structure in the substrate is not required for the reaction catalyzed by M1 RNA alone, but its presence markedly improves the efficiency of the reaction. In addition to its essential role in the biosynthesis of tRNA, RNAase P may have another function in vivo, namely, in the physiology of viral infections.

Base Sequence↗
PubMed1987

Model substrates for an RNA enzyme.

M1 RNA, the catalytic RNA subunit of Escherichia coli ribonuclease P, can cleave novel transfer RNA (tRNA) precursors that lack specific domains of the normal tRNA sequence. The smallest tRNA precursor that was cleaved efficiently retained only the domain of the amino acid acceptor stem and the T stem and loop. The importance of the 3' terminal CCA nucleotide residues in the processing of both novel and normal tRNA precursors implies that the same enzymatic function of M1 RNA is involved.

Base Sequence↗
PubMed1987

Suppressor and novel mutants of bacteriophage T4 tRNA(Gly).

We have isolated a weak UGA suppressor of phage T4 tRNA(Gly) in which the anticodon is changed from UCC to UCA. Two secondary mutants lacking suppressor activity are atypical in accumulating tRNA(Gly). Both mutations change the T stem of the cloverleaf model. One involved a G to A change at the 5' base position of the middle base-pair; the second involves a C to U change at a constant base position next to the T loop. The precursor RNAs of the mutants were cleaved in vitro with the catalytic RNA subunit of RNase P. Relative to normal precursor RNA, the precursor mutated at the middle base-pair position of the T stem was cleaved more rapidly, whereas the precursor mutated at the base-pair position next to the T loop was cleaved more slowly.

Base Sequence↗
PubMed1986

M1 RNA with large terminal deletions retains its catalytic activity.

Truncated transcripts of the rnpB gene from E. coli, coding for M1 RNA, the catalytic subunit of RNAase P, and fragments of M1 RNA generated by nuclease treatment have been prepared, and their ability to function catalytically in vitro has been determined. Molecules missing as many as 122 nucleotides at the 3' terminus retain catalytic activity, although at a much lower level than M1 RNA itself. No activity is observed with an RNA that is missing 70 nucleotides at the 5' terminus. The removal of even a small number of nucleotides from both termini eliminates all catalytic function. The preservation of one intact terminus may be essential for the tertiary and quaternary interactions required to generate the conformation of an active RNA species.

Bacterial Proteins↗
PubMed1986

Metal ion requirements and other aspects of the reaction catalyzed by M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli.

M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli, can under certain conditions catalytically cleave precursors to tRNA in the absence of C5, the protein moiety of RNase P. M1 RNA itself is not cleaved during the reaction, nor does it form any covalent bonds with its substrate. Only magnesium and, to a lesser extent, manganese ions can function at the catalytic center of M1 RNA. Several other ions either inhibit the binding of magnesium ion at the active site or function as structural counterions. The reaction rate of cleavage of precursors to tRNAs by M1 RNA is enhanced in the presence of poly-(ethylene glycol) or 2-methyl-2,4-pentanediol. Many aspects of the reaction catalyzed by M1 RNA are compatible with a mechanism in which phosphodiester bond cleavage is mediated by metal ion.

Binding Sites↗
PubMed1986

M1 RNA, the RNA subunit of Escherichia coli ribonuclease P, can undergo a pH-sensitive conformational change.

After purification from extracts of whole cells, M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli, apparently must undergo a change in conformation before it can function catalytically. The rate of this conformational change is dependent upon the duration of incubation at various temperatures and pH. delta E of the transition at pH 7.5 is approximately 36 kcal/mol. The change in conformation is not sensitive to Mg2+ concentration between 10 and 100 mM. A decrease in A260 of M1 RNA in solution has been observed during the incubation period that potentiates the conformational change at 30 degrees C, but no direct correlation can yet be made to specific structural rearrangements.

Endoribonucleases↗
PubMed1985

Characterization of an RNase P activity from HeLa cell mitochondria. Comparison with the cytosol RNase P activity.

A ribonuclease P-like activity was partially purified from HeLa cell mitochondria by DEAE-cellulose and octyl-Sepharose chromatography. RNase P-like activity can be quantitatively recovered from intact mitochondrial preparations treated with micrococcal nuclease, strongly suggesting that the enzyme is localized within the organelles. Mitochondrial RNase P (mtRNase P) cleaves the precursor to Escherichia coli suppressor tRNATyr at the same site as E. coli RNase P, producing the mature 5'-end of tRNATyr. The sensitivity of mtRNase P to pretreatment with nucleases or Pronase indicates that the enzyme has essential RNA and protein components. Although the ionic requirements of mtRNase P are similar to those of the RNase P activity isolated from the post-mitochondrial cytosol fraction, the chromatographic properties of mtRNase P are distinct. Mitochondrial RNase P is probably a part of the mitochondrial RNA processing machinery of mammalian mitochondria, being responsible for the endonucleolytic cleavage of the RNA transcripts at the 5'-side of the tRNA sequences.

Adenosine Triphosphate↗
PubMed1984

Structure in solution of M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli.

The structure of M1 RNA, the RNA component of Escherichia coli RNase P, has been probed by mild digestion with a variety of ribonucleases. The results have been used to generate a model for the two-dimensional structure of M1 RNA. This model is similar in many respects to an earlier model that was based entirely on theoretical considerations. M1 RNA was digested with RNase T1 in buffer containing 10 mM MgCl2 (in which M1 RNA, by itself, has no catalytic activity) and in buffer containing 60 mM MgCl2 (in which M1 RNA can cleave precursors to tRNA molecules). Under these conditions, the main features of the secondary structure are similar, but several minor differences are apparent. Such subtle changes in structure are also observed when M1 RNA is present in a binary complex with a substrate molecule, the precursor to E. coli tRNATyr.

Endoribonucleases↗
PubMed1984

Catalytic activity of an RNA molecule prepared by transcription in vitro.

Ribonuclease P is a ribonucleoprotein that cleaves precursors to transfer RNA (tRNA) molecules to yield the correct 5' terminal sequences of the mature tRNA's. The RNA moiety M1 RNA of ribonuclease P from Escherichia coli and the unprocessed transcript prepared in vitro of the gene for M1 RNA can both perform the cleavage reactions of the canonical enzyme in the absence of the protein moiety. When the transcript of the M1 RNA gene is combined with the protein moiety not only is a tRNA precursor cleaved but also the precursor to 4.5S RNA from Escherichia coli.

Base Sequence↗
PubMed1984

Cleavage of tRNA precursors by the RNA subunit of E. coli ribonuclease P (M1 RNA) is influenced by 3'-proximal CCA in the substrates.

tRNA precursor molecules that contain the CCA sequence found at the 3' termini of all mature tRNAs are cleaved in vitro more readily by M1 RNA, the catalytic subunit of E. coli RNAase P, than precursors that lack this sequence. The sensitivity to the CCA sequence is not apparent when precursors are cleaved by the reconstituted RNAase P holoenzyme that contains both M1 RNA and the protein subunit. These results have been obtained with monomeric precursor molecules encoded by the E. coli and human chromosomes and with three dimeric precursor molecules encoded by the bacteriophage T4 genome. The data are in agreement with previous results concerning T4 tRNA biosynthesis in vivo and show that the CCA sequence is important for the processing of precursors to tRNAs.

Base Sequence↗