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C Guerrier-Takada

Publications and source records attributed to C Guerrier-Takada.

35 records · Page 2Linked to original sources
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↗
PubMed1983

The activity of discrete fragments of ribosomal protein S1 in Q beta replicase function.

As a subunit of bacteriophage Q beta replicase, ribosomal protein S1 is required for tight binding of the enzyme to Q beta RNA and for the initiation of Q beta RNA transcription. To compare these properties of S1 with its functions in protein synthesis, we have reconstituted altered replicase enzymes by adding discrete fragments of S1 to Q beta replicase lacking S1 (R(-S1]. We show that the NH2-terminal region of S1 is required for S1 subunit interactions in replicase since a trypsin-resistant fragment (denoted S1-F1) lacking the NH2-terminal 31% of S1 is functionally inactive and does not seem to bind to R(-S1). Previous studies with S1-F1 indicated that this NH2-terminal region is required for S1 to bind to the ribosome. Our results also show that the COOH-terminal region of S1 is dispensable for S1's function in replicase because a mutant of S1 (m1-S1) lacking 21% of the COOH-terminal portion of the chain is as active as wild type S1 in replicase and binds to R(-S1) with comparable affinity. In protein synthesis, the mutant m1-S1 is known to substitute for S1 but is only about 75% as efficient as wild type S1.

Escherichia coli↗
PubMed1983

A G43 to U43 mutation in E. coli tRNAtyrsu3+ which affects processing by RNase P.

A novel mutation in the anticodon stem of E. coli tRNA1Tyrsu3+ (G43 to U43) has been characterized. The gene coding for the mutant tRNA, carried by phage phi 80DHA61.3 a derivative of phi 80psu3+su0, produces only 20% of mature suppressor tRNA as compared with phi 80psu3+. Both the mutant tRNA precursor and mature tRNA have an altered conformation. The precursor tRNA coded for by phi 80DHA61.3 is processed by RNase P more slowly than the su3+ precursor and does not form as stable an enzyme-substrate complex as does su3+ precursor. phi 80 DHA61.3 also contains a large deletion which begins in the spacer region between the su3+ gene and the su0- gene, extends through the su0- gene and includes most of the repeated region following the tRNA genes.

Anticodon↗
PubMed1983

The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme.

The RNA moieties of ribonuclease P purified from both E. coli (M1 RNA) and B. subtilis (P-RNA) can cleave tRNA precursor molecules in buffers containing either 60 mM Mg2+ or 10 mM Mg2+ plus 1 mM spermidine. The RNA acts as a true catalyst under these conditions whereas the protein moieties of the enzymes alone show no catalytic activity. However, in buffers containing 5-10 mM Mg2+ (in the absence of spermidine) both kinds of subunits are required for enzymatic activity, as shown previously. In the presence of low concentrations of Mg2+, in vitro, the RNA and protein subunits from one species can complement subunits from the other species in reconstitution experiments. When the precursor to E. coli 4.5S RNA is used as a substrate, only the enzyme complexes formed with M1 RNA from E. coli and the protein moieties from either bacterial species are active.

Bacillus subtilis↗
PubMed1982

Q beta replicase containing altered forms of ribosomal protein S1.

Transcription of Q beta RNA replicase requires that the host-encoded Escherichia coli ribosomal protein S1 be present as a subunit of the replicase. To determine whether the activities of S1 in protein synthesis are operational in Q beta RNA transcription, we formed altered replicase enzymes by reconstituting replicase lacking S1 (R(-S1)) with modified S1 species whose properties in nucleic acid binding and protein synthesis had been previously established. S1 derivatized with N-ethylmaleimide reconstitutes a modified replicase that is 81% as active as replicase reconstituted with unmodified S1. S1 derivatized with N-ethylmaleimide and unmodified S1 bind with comparable affinity to R(-S1) (1 X 10(8) M-1). These results indicate that the helix-unwinding properties of S1, which are known to be inactivated by N-ethylmaleimide modification, are not required for Q beta RNA transcription and that the sulfhydryl-derivatized region of S1 is not utilized in replicase subunit contacts. In contrast with its established ability to replace E. coli S1 on the ribosome, Caulobacter crescentus S1 does not reconstitute Q beta RNA transcription activity when added to R(-S1). Our results suggest this inactivity may be due to poor binding to R(-S1).

Coliphages↗
PubMed1982

Nucleotide sequence of the gene encoding the RNA subunit (M1 RNA) of ribonuclease P from Escherichia coli.

The gene encoding the RNA subunit (M1 RNA) of RNAase P (EC 3.1.26.5) from Escherichia coli has been isolated, and its complete nucleotide sequence, including flanking regions, has been determined. The promoter region, similar to others near genes under stringent control, and the site of transcription termination have been identified. The transcript from the gene (M1 RNA) can be drawn in a secondary structure that has approximately 60% G-C base pairs. One hairpin loop of this hypothetical structure has five contiguous nucleotides complementary to invariant nucleotides in the TpsiCG loop of all E. coli tRNAs. The M1 gene, when subcloned in the plasmid pBR325, can be amplified. It directs production of functional M1 RNA. In an E. coli strain thermosensitive for RNAase P function, the size of the gene transcript is the same as in wild-type E. coli, but less mature M1 RNA is made in the mutant cells.

Base Sequence↗
PubMed1981

Transfer RNA cross-linked to the elongation factor Tu subunit of Q beta replicase does not inhibit Q beta RNA replication.

One of the four subunits of bacteriophage Q beta RNA replicase is elongation factor Tu (EF-Tu), the host aminoacyl-tRNA (AA-tRNA) binding protein. To determine whether the RNA polymerase activity requires the tRNA binding site of EF-Tu, we reconstituted replicase with EF-Tu . GTP covalently bound to AA-tRNA. This cross-linked ternary complex (XLTC) was formed by the reaction of N epsilon-bromoacetyl-Lys-tRNA with EF-Tu-GTP. In an EF-Tu-dependent system for the reconstitution of replicase, XLTC restored polymerase activity at least as well as an equivalent amount of EF-Tu. Replicase reconstituted with XLTC was resolved from replicase containing EF-Tu by chromatography on phosphocellulose, a result which confirmed that the tRNA moiety was incorporated into the enzyme. Chromatographic analysis of reconstitution mixtures revealed that XLTC was incorporated into replicase as extensively as EF-Tu. From these results, it appears that the AA-tRNA binding site on EF-Tu is not required for the assembly or activity of Q beta RNA replicase. Furthermore, because the tRNA macromolecule is cross-linked to His-66 of the EF-Tu, the region surrounding His-66 must normally be exposed on the surface of the replicase.

Bacterial Proteins↗
PubMed1979

Size fractionation of DNA fragments by liquid-liquid chromatography.

A method for the fractionation of double-stranded DNA fragments from 150 to 22000 b.p. in size by liquid-liquid chromatography is described. The procedure makes use of the fact that the partitioning of DNA in a polyethylene glycol-dextran system is size dependent and can be altered by alkali metal cations. Cellulose or celite are used as supports for the stationary, dextran-rich phase. Examples show the fractionation of digests of T7 DNA produced by Dpn II and Hind II restriction endonulceases as well as lambda DNA digests produced by Hind III and Eco RI restriction endonucleases.

Bacteriophage lambda↗