Biomedical subjects

Yo Kikuchi

Publications and source records attributed to Yo Kikuchi.

At least 19 recordsLinked to original sources

PubMed2006

Characterization of extracellular RNAs produced by the marine photosynthetic bacterium Rhodovulum sulfidophilum.

The marine photosynthetic bacterium Rhodovulum sulfidophilum produces extracellular nucleic acids that are involved in its flocculation. These were found to be produced concomitantly with cell growth. The RNA fraction of these extracellular nucleic acids was subjected to cDNA analysis by applying a micro RNA cloning method and found to contain mainly fully mature-sized tRNAs and fragments of 16S and 23S rRNAs. Analyses of modified bases and genes of the RNAs revealed no structural difference between the intracellular and extracellular RNAs. This is the first report of structural analyses of bacterial extracellular RNAs.

Base Sequence↗

PubMed2005

Porphyrins and porphines bind strongly and specifically to tRNA, precursor tRNA and to M1 RNA and inhibit the ribonuclease P ribozyme reaction.

Porphyrins and porphines strongly inhibit the action of the RNA subunit of the Escherichia coli ribonuclease P (M1 RNA). Meso-tetrakis(N-methyl-pyridyl)porphine followed linear competitive kinetics with pre-tRNA(Gly1) from E. coli as variable substrate (Ki 0.960 microM). Protoporphyrin IX showed linear competitive inhibition versus pre-tRNA(Gly1) from E. coli (Ki 1.90 microM). Inhibition by meso-tetrakis[4-(trimethylammonio)phenyl]porphine versus pre-tRNA(Gly1) from E. coli followed non-competitive kinetics (Ki 4.1 microM). The porphyrins bound directly to E. coli tRNAVal, E. coli pre-tRNAGly1 and M1 RNA and dissociation constants for the 1:1 complexes were determined using fluorescence spectroscopy. Dissociation constants (microM) against E. coli tRNAVal and E. coli pre-tRNAGly were: meso-tetrakis(N-methyl-pyridyl)porphine 1.21 and 0.170; meso-tetrakis[4-(trimethylammonio)phenyl]porphine, 0.107 and 0.293; protoporphyrin IX, 0.138 and 0.0819. For M1 RNA, dissociation constants were 32.8 nM for meso-tetrakis(N-methyl-pyridyl)porphine and 59.8 nM for meso-tetrakis[4-(trimethylammonio)phenyl]porphine and excitation and emission spectra indicate a binding mode with strong pi-stacking of the porphine nucleus and base pairs in a rigid low-polarity environment. Part of the inhibition of ribonuclease P is from interaction with the pre-tRNA substrate, resulting from porphyrin binding to the D-loop/T-loop region which interfaces with M1 RNA during catalysis, and part from the porphyrin binding to the M1 RNA component.

Escherichia coli↗

PubMed2005

Substrate shape preference of Escherichia coli ribonuclease P ribozyme and holo enzyme using bottom-half part-shifting variants of pre-tRNA.

We showed previously that the bacterial ribonuclease P (RNase P) ribozyme has substrate shape preference depending on the concentrations of catalytically important magnesium ions. The ribozyme discriminates a canonical cloverleaf precursor tRNA from a hairpin RNA with a CCA-tag sequence at low concentrations of magnesium ions. By detailed analysis of the shape preference using the bottom-half part-shifting variants of a tRNA precursor, we showed that the RNAs in a T-shape structure can be substrates for the ribozyme reactions even at low concentrations of magnesium ions, and that the RNA in a natural L-shape is the best substrate for both the ribozyme and the holo enzyme. The results also showed that the position of the bottom-half part did not affect the cleavage site selection of a substrate by the enzyme. Our results are the first kinetic evidence to show the importance of the bottom-half part of tRNA molecule, and our result also showed that the holo enzyme can discriminate substrate shape as well as the ribozyme at low concentrations of metal ions.

Escherichia coli Proteins↗

PubMed2004

The P3 domain of E. coli ribonuclease P RNA can be truncated and replaced.

We prepared some truncated and replaced P3 mutants of Escherichia coli RNase P RNA, and used them to examine the RNase P ribozyme and holoenzyme reactions of a pre-tRNA substrate. The results indicated that mutations in the P3 domain did not affect the cleavage site selection of the pre-tRNA substrate, but did affect the efficiency of cleavage of the substrate. Results of stepwise truncation of the P3 domain and its replacement by the TAR sequence showed that the P3 domain of the E. coli RNase P was able to be truncated to certain length and was replaceable, but could not be deleted in the ribozyme.

Base Sequence↗

PubMed2004

Targeted isolation of a designated region of the Bacillus subtilis genome by recombinational transfer.

A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.

Bacillus subtilis↗

PubMed2004

Protein classification using comparative molecular interaction profile analysis system.

We recently introduced a new molecular description factor, interaction profile Factor (IPF) that is useful for evaluating molecular interactions. IPF is a data set of interaction energies calculated by the Comparative Molecular Interaction Profile Analysis system (CoMIPA). CoMIPA utilizes AutoDock 3.0 docking program, and the system has shown to be a powerful tool in clustering the interacting properties between small molecules and proteins. In this report, we describe the application of CoMIPA for protein clustering. A sample set of 15 proteins that share less than 20% homology and have no common functional motifs in primary structure were chosen. Using CoMIPA, we were able to cluster proteins that bound to the same small molecule. Other structural homology-based clustering programs such as PSI-BLAST or PFAM were unable to achieve the same classification. The results are striking because it is difficult to find any common features in the active sites of these proteins that share the same ligand. CoMIPA adds new dimensions for protein classification and has the potential to be a helpful tool in predicting and analyzing molecular interactions.

Algorithms↗

PubMed2004

Region dependent efficiency for recombinational transfer of the Bacillus subtilis 168 genome.

Submega-sized regions of the Bacillus subtilis genome were cloned to plasmid by the B. subtilis Recombinational Transfer (BReT) method. BReT efficiency depends not only on the genome location but also on the choice of sequences for simultaneous homologous recombination during BReT. In an extreme case, a 91-kb region that was unsuccessful on the first attempt was obtained when the slightly shifted 98-kb region was targeted.

Bacillus subtilis↗

PubMed2004

Examining the bases of the J3/4 domain of Escherichia coli ribonuclease P.

We prepared several mutants of the J3/4 and P4 domains of Escherichia coli ribonuclease P (RNase P): A62G, A62U, G63C/G64C, A65G, A67G, U69A, U69G, U69C, U69Delta, and U69UU. Comparison of the ribozyme and holo enzyme reactions at various concentrations of magnesium ions showed that the presence of a bulge at U69 in the P4 domain was important in the holo enzyme. The results also showed that the conserved bases G63 and G64 in the J3/4 domain were important for efficient ribozyme reactions but were replaceable in the presence of the protein component. Our data showed that the bases in the J3/4 and P4 domains displayed different responses to the metal ions that were affected by the presence of the protein component.

Base Sequence↗

PubMed2004

The natural intron sequence of human tyrosine pre-transfer RNA is not a temporal stabilizer for cloverleaf structure.

We have developed the hyperprocessing technique to evaluate the stability of the cloverleaf shape of pre-transfer RNA (pre-tRNA). Application of this strategy to hyperprocessible human tyrosine pre-tRNA indicated that the natural intron sequence did not contribute to stabilization of the cloverleaf shape of this pre-tRNA, while the artificial intron with elongated anticodon-stem completely inhibited hyperprocessing of it. Our data suggested that the contemporary intron sequence may be a vestige of the ancient pre-biotic world, but not has been retained as a temporal stabilizer of the pre-tRNA before the base modifications.

Base Sequence↗

PubMed2004

Mutational analysis of the length of the J3/4 domain of Escherichia coli ribonuclease P ribozyme.

We prepared a series of length variants of the J3/4 domain of Escherichia coli ribonuclease P (RNase P) ribozyme: the four-base long J3/4 domain (A(62)G(63)G(64)A(65)) was replaced with GGA (denoted DeltaA), GA (DeltaAG), A (DeltaAGG), AAGGA (SigmaA), AAAGGA (SigmaAA), and AAAAGGA (SigmaAAA). The results indicated that truncating and inserting operations of the J3/4 domain drastically reduced ribozyme activity (WT>>SigmaAA>SigmaA>SigmaAAA>>DeltaAG>DeltaA, DeltaAGG), but did not affect the cleavage site selection of a substrate by the ribozyme. The reduced ribozyme activity of each mutant was rescued to some extent by the addition of a high concentration of magnesium ions. Our data indicate that the conserved AGGA sequence was important for efficient ribozyme reactions, and suggested that the length mutations affected ribozyme activity through metal ion binding steps.

Catalysis↗

PubMed2003

Molecular evaluation using in silico protein interaction profiles.

MOTIVATION: To find a correlation between the activities and structures of molecules is one of the most important subjects for molecular evaluation study. Traditional quantitative structure-activity relationship (QSAR) methodologies represent those attempts using physicochemical descriptors. Creating a new molecular description factor based on the results of a computational docking study will add new dimensions to molecular evaluation. RESULTS: We propose a new molecular description factor analysis system called the Comparative Molecular Interaction Profile Analysis (CoMIPA) system in which the AutoDock program is used for docking evaluation of small molecule compound-protein complexes. Interaction energies are calculated, and the data sets obtained are called interaction profiles (IPFs). Using the IPF as a scoring indicator, the system could be a powerful tool to cluster the interacting properties between small molecules and bio macromolecules such as ligand-receptor bindings. Further development of the system will enable us to predict the adverse effects of a drug candidate.

Algorithms↗

PubMed2003

Substrate shape specificity of E coli RNase P ribozyme is dependent on the concentration of magnesium ion.

The bacterial RNase P ribozyme can accept a hairpin RNA with CCA-3' tag sequence as well as a cloverleaf pre-tRNA as substrate in vitro, but the details are not known. By switching tRNA structure using an antisense guide DNA technique, we examined the Escherichia coli RNase P ribozyme specificity for substrate RNA of a given shape. Analysis of the RNase P reaction with various concentrations of magnesium ion revealed that the ribozyme cleaved only the cloverleaf RNA at below 10 mM magnesium ion. At 10 mM magnesium ion or more, the ribozyme also cleaved a hairpin RNA with a CCA-3' tag sequence. At above 20 mM magnesium ion, cleavage site wobbling by the enzyme in tRNA-derived hairpin occurred, and the substrate specificity of the enzyme became broader. Additional studies using another hairpin substrate demonstrated the same tendency. Our data strongly suggest that raising the concentration of metal ion induces a conformational change in the RNA enzyme.

Base Sequence↗

PubMed2003

Revisiting the substrate recognition of bacterial ribonuclease P: in the view of the recognition of the base N73 in the substrate.

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. At high concentration of Mg ion, the substrate shape preference of the ribozyme becomes broader to accept a hairpin shape RNA. In hairpin RNA cleavage reactions, we found that the base interaction between the base U294 of E. coli ribozyme and the base N73 of the substrate RNA did not obey the response according to the Watson-Crick type interaction which is usually observed in the interaction between the base U294 of ribozyme and the base N73 of tRNA precursor.

Base Pairing↗

PubMed2003

Extracellular DNA and RNA produced by a marine photosynthetic bacterium Rhodovulum sulfidophilum.

A marine photosynthetic bacterium Rhodovulum sulfidophilum secretes nucleic acids that are involved in flocculating ability. These extracellular nucleic acids have not been well characterized. Here, we have analyzed these nucleic acids and revealed that the extracellular nucleic acids are a mixture of double-stranded DNAs and single-stranded RNAs. The DNAs have sizes of more than 30 kbp and at least a part of these DNAs is probably an amplified portion of genomic DNA. The RNAs seems to be tRNA like molecules from size estimation.

DNA, Bacterial↗

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↗