Biomedical subjects

Yo Kikuchi

Publications and source records attributed to Yo Kikuchi.

29 records · Page 2Linked to original sources

PubMed2003

Recognition of tRNA bottom half by bacterial ribonuclease P.

Bacterial ribonuclease P (RNase P) contains a catalytic RNA that cleaves precursor tRNA to form the 5'-end of mature tRNA. Bacterial RNase P mainly recognizes the acceptor stem and T arm modules of tRNA molecules. The region consisting of T arm, acceptor stem and 3' CCA motif in the tRNA is generally termed "top half", and the region consisting of the others, anticodon arm, extra loop and D arm, is called "bottom half". The stems in the top half contribute to recognition, but effects of the bottom half have not been elucidated. To study the effects of the bottom half on the RNase P recognition, we have synthesized several mutant substrates that have the bottom half on different positions along the top half stem. Most of these mutants were cleaved by Escherichia coli RNase P precisely at the expected position, but the cleavage efficiencies were very different especially at low Mg2+ concentration. We also found that RNase P holoenzyme prefered somewhat mutated tRNA precursor to the wild-type tRNA precursor.

Animals↗

PubMed2003

Bacterial ribonuclease P reaction is affected by substrate shape and magnesium ion concentration.

Bacterial RNase P is a ribonucleoprotein enzyme which cleaves 5'-precursor sequence of pre-tRNA for pre-tRNA maturation. The RNA component of bacterial RNase P is ribozyme. It recognizes cloverleaf shaped pre-tRNA and hairpin RNA with a CCA-3' tag sequence as its substrates. Previously, we reported that the substrate recognition of the E. coli RNase P ribozyme depends on the concentration of magnesium ion in vitro. In this report, we examined the substrate shape preference of the Bacillus subtilis RNase P ribozyme and compared it with that of the E. coli ribozyme. The results of the B. subtilis ribozyme displayed same tendency as the E. coli ribozyme. We also examined the effect of the protein component of the E. coli RNase P. Under the conditions tested, magnesium ion concentration dependency to substrate shape recognition was not observed when the holo enzyme was used.

Bacillus subtilis↗

PubMed2003

Application of recombination transfer to the cognate Bacillus subtilis genome.

Isolation of the designated genome region of Bacillus subtilis was investigated using a B. subtilis recombinational transfer (BReT) system. Two DNA sequences flanking the precise genome region are cloned in the BReT vector. The BReT plasmid recovered the predicted genome sequence as large as 100 kb with high fidelity. The result indicates that the BReT system originally developed to recover the non-cognate segments cloned in the B. subtilis genome vector can be applied to the cognate sequence.

Bacillus subtilis↗

PubMed2003

Escherichia coli tRNAs are resistant to the hyperprocessing reaction of homologous E. coli ribonuclease P ribozyme.

Bacterial ribonuclease P RNA ribozyme can do the hyperprocessing reaction, the internal cleavage reaction of some floppy eukaryotic tRNAs. The hyperprocessing reaction can be used as a detection tool to examine the stability of the cloverleaf shape of tRNA. Until now, the hyperprocessing reaction has been observed in the heterologous combination of eukaryotic tRNAs and bacterial RNase P enzymes. In this paper, we examined the hyperprocessing reaction of Escherichia coli tRNAs by homologous E. coli RNase P, to find that these homologous tRNAs were resistant to the toxic hyperprocessing reaction. Our results display the evidence for molecular co-evolution between homologous tRNAs and RNase P in the bacterium E. coli.

Bacillus subtilis↗

PubMed2003

Comparative analyses of hairpin substrate recognition by Escherichia coli and Bacillus subtilis ribonuclease P ribozymes.

Previously, we reported that the substrate shape recognition of the Escherichia coli ribonuclease (RNase) P ribozyme depends on the concentration of magnesium ion in vitro. We additionally examined the Bacillus subtilis RNase P ribozyme and found that the B. subtilis enzyme also required high magnesium ion, above 10 mM, for cleavage of a hairpin substrate. The results of kinetic studies showed that the metal ion concentration affected both the catalysis and the affinity of the ribozymes toward a hairpin RNA substrate.

Bacillus subtilis↗

PubMed2003

The protein component of bacterial ribonuclease P flickers the metal ion response to the substrate shape preference of the ribozyme.

The substrate shape specificity of the Escherichia coli ribonuclease P (RNase P) ribozyme depends on the concentration of magnesium ion. At 10 mM or more, it can cleave a hairpin substrate as well as a cloverleaf pre-transfer RNA (tRNA). The results showed, however, that the holo enzyme cleaved the hairpin substrate at low concentrations of magnesium ion. Considering that the homologous E. coli tRNAs are resistant to internal cleavage by the RNase P, the phenomena suggest that this catalytic activity might take part in the removing the mis-folded RNAs in the cell.

Catalysis↗

PubMed2002

Guide DNA technique in bacterial ribonuclease P reaction for effective processing of tRNA precursor.

Previously, we found that a small (approx. 20-mer) DNA hybridizing to the 5'-leader region of a tRNA precursor enhances the cleavage efficiency in bacterial ribonuclease P reaction. We named this technique the 'guide DNA technique'. Detailed analyses showed that the length of the guide DNA, concentration of the guide DNA and the hybridizing position affected the cleavage efficiency: for an effective cleavage reaction, guide DNA should be designed to hybridize to the region on the cleavage site, should be 20 bases or more in length and should be of high concentration. The presence of a 5'-flanking region in the DNA did not affect the cleavage reaction. The guide DNA technique is a useful tool for effective preparation of mature tRNA molecules in vitro.

Animals↗

PubMed2002

Porphyrins and porphines inhibit the ribonuclease P reaction in vitro.

Porphyrin has been reported to bind to the T psi C stem of tRNA. This site is also recognized by ribonuclease P, which is essential and ubiquitous endoribonuclease responsible for the maturation of 5' ends of tRNA precursors. Thus, we investigated the effects of porphyrins on the in vitro reaction of ribonuclease P from Escherichia coli. The results showed that some of porphyrins inhibited the reaction more strongly than any other inhibitors reported so far. In addition to the benzimidazole inhibition that we have previously reported, these unusual substrate-binding inhibitions may provide new leads for the novel anti-bacterial reagent design.

Endoribonucleases↗

PubMed2002

Regulation of bacterial RNase P ribozyme reaction by divalent cation and guide DNA.

The RNA subunit of bacterial ribonuclease P (RNase P) is a ribozyme which can cleave a canonical cloverleaf tRNA precursor and a hairpin RNA with a CCA-3' tag sequence as its substrate. With high concentration of Mg ion, the ribozyme as well as holo enzyme internally cleaves certain tRNAs in vitro. We denoted this unusual reaction as hyperprocessing. By controlling magnesium ion concentration for the reaction and also by forcing the RNA shape with external guide DNAs, we could regulate the hyperprocessing reaction by the bacterial RNase P enzymes. These techniques will lead the RNase P ribozyme to more designable and more applicable RNA-cleaving enzyme.

Bacteria↗

PubMed2002

Another cut for lysine tRNA: application of the hyperprocessing reaction reveals another stabilization strategy in metazoan lysine tRNAs.

Recently, we revealed that the cloverleaf structure of some eukaryotic tRNAs is not always stable in vitro, and the denatured structures of these tRNAs are sometimes detected in bacterial RNase P reactions. We have designated the unusual internal cleavage reaction of these tRNAs as hyperprocessing. We have developed this hyperprocessing strategy as a useful tool for examining the stability of the tRNA cloverleaf structure. There are some common features in such unstable, hyperprocessible tRNAs, and the criteria for the hyperprocessing reaction of tRNA are extracted. Metazoan initiator methionine tRNAs and lysine tRNAs commonly fit the criteria, and are predicted to be hyperprocessible. The RNase P reactions of two metazoan lysine tRNAs from Homo sapiens and Caenorhabditis elegans, which fit the criteria, resulted in resistance to the internal cleavage reaction, while one bacterial lysine tRNA from Acholeplasma laidlawii, which also fits the criteria, was internally cleaved by the RNase P. The results showed that the metazoan lysine tRNAs examined are very stable without base modifications even under in vitro conditions. We also examined the 3'-half short construct of the human lysine tRNA, and the results showed that this RNA was internally cleaved by the enzyme. The results indicated that the human lysine tRNA has the ability to be hyperprocessed but is structurally stabilized in spite of lacking base modifications. A comparative study suggested, moreover, that the acceptor-stem bases should take part in the stabilization of metazoan lysine tRNAs. Our data strongly suggest that the cloverleaf shape of other metazoan lysine tRNAs should also be stabilized by means of similar strategies to in the case of human tRNA(Lys3).

Animals↗

PubMed2002

Kinetics of hyperprocessing reaction of human tyrosine tRNA by ribonuclease P ribozyme from Escherichia coli.

Human tyrosine tRNA and fly alanine, histidine, and initiator methionine tRNAs are generally cleavable internally by bacterial ribonuclease P ribozyme. The unusual internal cleavage reaction of tRNA, called hyperprocessing, occurs when the cloverleaf structure of the tRNA molecule is denatured to form a double-hair-pin-like structure. The hyperprocessing reaction of these tRNAs requires magnesium ions. We analyzed details of this reaction using human tyrosine tRNA and Escherichia coli RNase P ribozyme. The usual processing reaction occurred efficiently with magnesium at 5 mM, but for the hyperprpocessing reaction, higher concentrations were needed. With such high concentrations, hyperprocessing cleaved both mature tRNA and tRNA precursor as substrates. When mature tRNA was the substrate, the apparent K(M) was almost the same as in the usual reaction, but k(cat) was smaller. These results indicated that the occurrence of hyperprocessing depends on the magnesium ion concentration, and suggested that magnesium ions contribute to the recognition of the shape of the substrate by bacterial RNase P enzymes.

Base Sequence↗