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E Ohtsuka

Publications and source records attributed to E Ohtsuka.

At least 199 records · Page 11Linked to original sources
PubMed1988

Increase in nucleolytic activity of ribonuclease T1 by substitution of tryptophan 45 for tyrosine 45.

The preparation and analysis of a mutant ribonuclease (RNase) T1 which possesses higher nucleolytic activity than the wild-type enzyme are described. The gene for the mutant RNase T1 (Tyr45----Trp45), in which a single amino acid at the binding site of the guanine base has been changed, was constructed by the cassette mutangenesis method using a chemically synthesized gene [Ikehara, M. et al. (1986) Proc. Natl Acad. Sci. USA 83, 4695-4699]. In order to reduce the nucleolytic activity of the enzyme in vivo, this gene was expressed in Escherichia coli as a fused protein connected through methionine residues to other proteins at both the N- and C-termini. After liberation from the fused protein by cleavage with cyanogen bromide at the methionine junctions, the mutant RNase T1 was purified by column chromatography. The nucleolytic activity toward pGpC increased to 120% of that of wild-type RNase T1. The kinetic parameters of the mutant enzyme demonstrate that this higher nucleolytic activity is due to a higher affinity for the substrate, probably because of an increased stacking effect in the binding pocket for the guanine base. This mutant enzyme also possessed a higher nucleolytic activity against pApC than wild-type RNase T1.

Base Sequence↗
PubMed1988

Crystallization of human c-H-ras oncogene products.

There is compelling evidence that cancer develops as a consequence of genetic changes (probably multiple) in some members of a selected set of cellular genes. DNA isolated from a variety of tumors, but not normal tissues, possesses the ability to malignantly transform non-tumorigenic cells. Many oncogenes responsible for such transformation have been isolated from transformed cell lines and animal and human tumors induced spontaneously, by virus, by chemical, or by radiation. The most commonly found transforming genes isolated from human tumor cells by DNA transfection assay are the ras gene family (c-H-ras, c-K-ras and N-ras). We report crystallization of several human c-H-ras oncogene proteins.

Crystallization↗
PubMed1988

Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21.

The crystal structure at 2.7 A resolution of the normal human c-H-ras oncogene protein lacking a flexible carboxyl-terminal 18 residue reveals that the protein consists of a six-stranded beta sheet, four alpha helices, and nine connecting loops. Four loops are involved in interactions with bound guanosine diphosphate: one with the phosphates, another with the ribose, and two with the guanine base. Most of the transforming proteins (in vivo and in vitro) have single amino acid substitutions at one of a few key positions in three of these four loops plus one additional loop. The biological functions of the remaining five loops and other exposed regions are at present unknown. However, one loop corresponds to the binding site for a neutralizing monoclonal antibody and another to a putative "effector region"; mutations in the latter region do not alter guanine nucleotide binding or guanosine triphosphatase activity but they do reduce the transforming activity of activated proteins. The data provide a structural basis for understanding the known biochemical properties of normal as well as activated ras oncogene proteins and indicate additional regions in the molecule that may possibly participate in other cellular functions.

Amino Acid Sequence↗
PubMed1988

Construction of a series of several self-cleaving RNA duplexes using synthetic 21-mers.

Two fragments (21-mers) containing consensus sequences for the self-cleavage domain in transcripts of satellite DNA of the newt were chemically synthesized and found to be cleaved in the presence of Mg2+. The cleaved product contained the 3'-terminal 2',3'-cyclic phosphate. Twenty-five combinations of partially double-stranded 21-mer RNA which contained different bases within the consensus sequences and at the cleavage sites were tested for self-cleavage. It seemed that guanosine 3'-phosphate was not susceptible to transesterification at the cleavage site.

Animals↗
PubMed1988

Glu 46 of ribonuclease T1 is an essential residue for the recognition of guanine base.

The Glu 46 of ribonuclease T1, which is assumed to interact with Nl of the guanine residue in RNA by a hydrogen bond from the result of X-ray analysis, was changed to alanine by site-directed mutagenesis and its function examined. The nucleolytic activity of the Ala 46 mutant enzyme against pGpC decreased to 0.4% of that of the wild-type enzyme, on the other hand its activity against pApC increased. This result suggests that the Glu 46 is essential for the recognition of the guanine base but that it also interferes with the recognition of the adenine base.

Alanine↗
PubMed1988

Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering.

Recognition by ribonuclease T1 of guanine bases via multidentate hydrogen bonding and stacking interactions appears to be mediated mainly by a short peptide segment formed by one stretch of a heptapeptide, Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47- Phe48. The segment displays a unique folding of the polypeptide chain--consisting of a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by a hydrogen-bond network involving the side chain of Asn44, the main-chain atoms of Asn44, Gly47 and Phe48 and one water molecule. The segment is connected to the C terminus of a beta-strand and expands into a loop region between Asn43 and Ser54. Low values for the crystallographic thermal parameters of the segment indicate that the structure has a rigidity comparable to that of a beta-pleated sheet. Replacement of Asn44 with alanine leads to a far lower enzymatic activity and demonstrates that the side chain of Asn44 plays a key role in polypeptide folding in addition to a role in maintaining the segment structure. Substitution of Asn43 by alanine to remove a weak hydrogen bond to the guanine base destabilized the transition state of the complex by 6.3 kJ/mol at 37 degrees C. In contrast, mutation of Glu46 to alanine to remove a strong hydrogen bond to the guanine base caused a destabilization of the complex by 14.0 kJ/mol. A double-mutant enzyme with substitutions of Asn43 by a histidine and Asn44 by an aspartic acid, to reproduce the natural substitutions found in ribonuclease Ms, showed an activity and base specificity similar to that of the wild-type ribonuclease Ms. The segment therefore appears to be well conserved in several fungal ribonucleases.

Amino Acid Sequence↗
PubMed1988

Conversion of the guanine nucleotide binding sites of ras protein resulting in the reduction of base specificity.

A gene coding for the novel ras protein, p21X, in which the domains of guanine binding and phosphate binding were exchanged, was constructed and expressed in Escherichia coli. The gene product, p21X, showed GTP binding activity, but no GPTase activity. In addition, p21X revealed binding activity toward ATP and CTP. In a competitive binding assay, [3H]GTP binding to p21X was inhibited in the presence of ATP, CTP and UTP, ITP as well as GDP, GTP and dGTP.

Amino Acid Sequence↗
PubMed1988

Sequence-specific cleavage of RNA using chimeric DNA splints and RNase H.

To cleave RNA molecules using E. coli RNase H in a site-specific manner, a short oligodeoxyribonucleotide (3-5 mer) linked with oligo(2'-O-methyl)ribonucleotide(s) was designed to be used as a DNA splint. Our model experiments with ribooligomer the splint duplexes (9 mers) and RNase H demonstrated that a tetradeoxynucleotide cluster seems to be sufficient for the enzyme recognition and the short DNA-containing splint directs a unique cleavage of RNA by RNase H. The method could be applied to longer ribooligonucleotide substrates. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.

Base Sequence↗
PubMed1987

Two histidine residues are essential for ribonuclease T1 activity as is the case for ribonuclease A.

Ribonuclease T1 (RNase T1, EC 3.1.27.3) is a guanosine-specific ribonuclease that cleaves the 3',5'-phosphodiester linkage of single-stranded RNA. It is assumed that the reaction is generated by concerted acid-base catalysis between residues Glu-58 and His-92 or His-40. From the results of chemical modification and NMR studies, it appeared that the residue Glu-58 was indispensable for nucleolytic activity. However, we have recently demonstrated that Glu-58 is an important but not an essential residue for catalytic activity, using the methods of genetic engineering to change Glu-58 to Gln-58 etc [Nishikawa, S., Morioka, H., Fuchimura, K., Tanaka, T., Uesugi, S., Ohtsuka, E., & Ikehara, M. (1986) Biochem. Biophys. Res. Commun. 138, 789-794]. In the present paper, we report that mutants of RNase T1 with residue Ala-40 or Ala-92 have almost no activity, while mutants that contain Ala-58 retain considerable activity. These results show that the two histidine residues, His-40 and His-92, but not Glu-58, are indispensable for the catalytic activity of the enzyme. We propose a revised reaction mechanism in which two histidine residues play a major role, as they do in the case of RNase A.

Aspergillus oryzae↗
PubMed1987

Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides.

2'-O-Methyl derivatives of the common ribonucleosides except for guanosine were synthesized via the 2'-O-methylation of appropriately-protected nucleosides with CH3I in the presence of Ag2O. The 2'-O-methylguanosine derivative was prepared by the monomethylation of a 2',3'-cis-diol system with diazomethane. These derivatives were converted to protected 2'-O-methylribonucleoside 3'-phosphates and used for oligonucleotide synthesis on polymer supports. Thus, oligo(2'-O-methyl-ribonucleotides) having the sequence identical to the consensus sequence of the 5'-splice junction CAGGUAAGU and its complement were synthesized in a stepwise manner using the phosphotriester method. Thermal stabilities (Tm's) of the duplex of these 2'-O-methyl ribo-oligomers and eight related duplexes containing ribo- or deoxyribo-oligomers were examined. It was found that the 2'-O-methyl oligoribonucleotides can be utilized as an alternative to an oligoribonucleotide probe in RNA hybridizations as the hybrid formed has a high, or a higher Tm, the probe is much easier to synthesize and it is less likely to be enzymatically degraded.

Acetylation↗
PubMed1987

The in vivo stability, maturation and aminoacylation of anticodon-substituted Escherichia coli initiator methionine tRNAs.

We have constructed eight anticodon-modified Escherichia coli initiator methionine (fMet) tRNAs by insertion of synthetic ribotrinucleotides between two fragments ('half molecules') derived from the initiator tRNA. The trinucleotides, namely CAU (the normal anticodon), CAA, CAC, CAG, GAA, GAC, GAG and GAU, were joined to the 5' and 3' tRNA fragments with T4 RNA ligase. The strategy of reconstruction permitted the insertion of radioactive 32P label between nucleotides 36 and 37. tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes, and the following properties were evaluated: the stability of these eubacterial tRNA variants in the eukaryotic oocytes; the enzymatic modification of the adenosine at position 37 (3' adjacent to the anticodon) and aminoacylation of the chimeric tRNAs by endogenous oocyte aminoacyl-tRNA synthetases. In contrast to other variants, the two RNAs having CAU and GAU anticodons were stable and underwent quantitative modification at A-37. These results show that the enzyme responsible for the modification of A-37 to N-[N-(9-beta-D-ribofuranosylpurine-6-yl)carbamoyl]threonine (t6A) is present in the cytoplasm of oocytes and is very sensitive to the anticodon environment of the tRNA. Also, these same GAU and CAU anticodon-containing tRNAs are fully aminoacylated with the heterologous oocyte aminoacyl-tRNA synthetases in vivo. During the course of this work we developed a generally applicable assay for the aminoacylation of femtomole amounts of labelled tRNAs.

Animals↗
PubMed1987

Site-directed cleavage of RNA.

Using complementary chimeric oligonucleotides containing deoxyribonucleotides and 2'-O-methylribonucleotides (1), enzymatically synthesized RNA (90 mer) were cleaved at a single site with Escherichia coli RNaseH, either at a hairpin loop or at a stem region. Especially, site-specific cleavage occurred in even the target region being enclosed within a stable, base-paired stem. The method is proved to be generally applicable to RNA containing secondary structures.

Base Sequence↗
PubMed1987

Identification of I:A mismatch base-pairing structure in DNA.

Deoxyoligonucleotides containing deoxyinosine residues at positions corresponding to ambiguous nucleotides derived from an amino acid sequence have been successfully used as hybridization probes. It is assumed that the hypoxanthine residue can make base pairs with multiple bases. In order to obtain direct evidence for I:A base-pairing, a self-complementary deoxyoligonucleotide, d(G-G-I-A-C-C), was synthesized and its properties were examined by NMR spectroscopy. Three hydrogen-bonded imino proton resonances are observed at low temperatures in H2O suggesting the formation of a self-duplex with complete base pairing. Nuclear Overhauser effect (NOE) experiments showed that a signal at 15.1 ppm originated from the imino proton (H1) of the dI residue (I3) which is hydrogen-bonded to the dA residue (A4). Both the I3 and A4 residues were assumed to have taken an anti glycosidic conformation since irradiating the H1 of I3 gave NOEs both to its own H2 and to that of A4, an NOE also being observed between the H2 protons of I3 and A4. Comparison of the 31P NMR spectra of d(G-G-I-A-C-C) and d(G-G-I-C-C-C) showed the backbone structure of d(G-G-I-A-C-C) to have been disturbed by the presence of purine:purine base pairs in the middle of the hexamer duplex.

Base Composition↗
PubMed1987

Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H.

We found that, in the presence of chimeric oligonucleotides containing complementary deoxyribo- and 2'-O-methylnucleosides, a nonaribonucleotide, [5'-32P]pACUUACCUG, was cleaved specifically upon treatment with RNase H. When 3'm(UG)d(AATG)m(GAC)5' was used as a hybridization strand, pACUUACCUG was cleaved between C6 and C7 to yield pACUUAC. In the presence of 3'm(UGAA)d(TGGA)m(C)5', the nonaribonucleotide was hydrolyzed, mainly between U8 and C9, to give pACUUACCU. This method will have a variety of applications in the field of RNA engineering.

Autoradiography↗