Shosuke Takemura

Crystallization and preliminary X-ray diffraction study of 5 S rRNA from Thermus thermophilus HB8

5 S rRNA is a common ribosomal component which is present in various species from prokaryotes to eukaryotes. Many attempts were made to elucidate its structure and function [ 11. A number of the models for its secondary structure including that by Nishikawa and Takemura have been proposed [ 11. The best way for elucidating a definite threedimensional structure of a macromolecule is to prepare its single crystal and analyze it by X-ray crystallography. Crystallization is thus a significant step of the analysis. 5 9 rRNA from extremely thermophilic bacteria may be more suitable for crystallization, since it is supposed to have a more stable structure than those from usual non-thermophilic bacteria. We describe here that the RNA purified from Thermus thermophilus HB8 can be certainly crystallized. 5 S rRNA is the second natural nucleic acid that has been obtained as a single crystal. The nucleotide sequence of the same RNA as used for the crystallization has been recently reported by Kumagai et al. [2].

Recovery of tyrosine acceptor activity by combining 3′-half molecule with stepwise degradation products of 5′-half molecule obtained from tyrosine tRNA

Abstract Two half molecules were obtained from purified Torulopsis utilis tyrosine tRNA by scission with RNase T1 of the G-ψ bond of the presumed anticodon GψA. When these halves were mixed, full tyrosine acceptor activity was recovered, although each of the halves was inactive. After splitting the 3′-terminal residues of the 5′-half and mixing the products with the 3′-half, the activity was fully recovered as compared with native tyrosine tRNA, clearly indicating that the first letter of the anticodon is not regarded as one of the tyrosine recognition sites.

Nucleotide sequence of tRNAGly 2 from the posterior silk glands of Bombyx mori

The posterior silk glands of Born&x mori exclusively produce fibroin from day 5-8 of the 5th instar. In proportion to the amino acid content of Bbroin, this organ synthesizes much larger amounts of tRNAs specific for glycine, alanine and serine than those specific for the other amino acids [ 1,2] . Because of the characteristic synthesis and accumulation of the particular tRNA species, the silk glands should be promising as a system to study on the correlation of tRNA and mRNA syntheses. In spite of an interest to the function of the posterior silk glands, sequencing of tRNA from this organ was scarcely carried out so far. Glycine tRNA from the posterior silk glands is composed of two major species, i.e., tRNA?‘y and tRNAflY [3] *. We present here the primary structure of the tRNA?lY .

Nucleotide sequence of alanine tRNA I from Torulopsis utilis

Nucleotide sequences of Torulopsis utilis tRNAyd*, tRNAne and tRNATvr (all the major molecular species) have been determined in our laboratory [ l--3] . T.utilis is often called torula yeast but belongs to a taxonomically different “class” from baker’s or brewer’s yeasts (Sacchurom_r~~s ccrevisiue); the former is classified into Furzgi imperfecti and the latter into Ascomycetes. Such a difference of species reflects in the primary structures of tRNA’s, and thus T.utilis tRNATvr and tRNAp1 differ in several nucleotides from the corresponding Xcerevisiue tRNA’s [3,4] . However these and several other tRNA’s from T.utilis so far assayed have been fully aminoacylated with the corresponding aminoacyl-tRNA synthetases from baker’s yeast. Sequence homology between T.utilis and E.ccoli tRNA’sIc[2,5] is less than that between the above T.utilis and Xcerevisiae tRNA’s, and approximately half of either tRNAne can be aminoacylated with the heterologous isoleucyl-tRNA synthetase**. A comparison of the sequences of tRNA’s of different species but of identical amino acid specificity, and kinetics of the heterologous aminoacylation will offer useful information on the aminoacyl-tRNA synthetase recognition sites in tRNA. Alanine tRNA of T.utilis can be also fully acylated with Scerevisiae alanyl-tRNA synthetase. We would like to report the established sequence of the major species of T.utilis ahnine tRN.4 CtRNAt’) and structural differences

A comparison between inosine- and guanosine-containing anticodons in ribosome-free codon-anticodon binding

Abstract Binding of the ribooligomers, AUC, AUU, AUA, AUCA, AUUA, and AUAA to the isoleucine-accepting tRNAs, tRNA T. utilis Ile (anticodon, -IAU-) and tRNA E. coli Ile (anticodon, -GAU-) was measured by equilibrium dialysis. With the aid of Scatchard plots, AUCA was shown to bind to one site per tRNA molecule, presumably the anticodon. AUA and AUAA did not measurably attach to either anticodon in the ribosome-free system. All other oligomers were bound to tRNA E. coli Ile about 5 to 13 times stronger than to tRNA T. utilis Ile .

Adenylic acid-specific limited digestion of tRNA with ribonuclease U2 and the C-C-A terminal repair of resulting fragments

1. Limited digestion of Torulopsis utilis tRNA1Val with ribonuclease U2 resulted in preferential cleavages at the A-C bond adjacent to the C-C-A terminus, and at two A-C bonds and partially at an I-A bond in the anticodon loop. Consequently three large fragments were obtained: 5′-half, C-C-A-depleted 3′-half and C-C-A-depleted tRNA. 2. Adenylic acid incorporation into those fragments was studied in the presence of tRNA nucleotidyltransferase. The combination of 5′- and 3′-half-molecules incorporated 63 % of the theoretical amount of adenylic acid. No incorporation was observed in the case of the 5′-moiety alone, though the 3′-half-molecule alone showed about 20 % incorporation. 3. When the valine-acceptor activities of those fragments were assayed, no activity was evidenced with the combination of 5′- and 3′-half-molecules, while the C-C-A-depleted fragment showed 17 % of the valine acceptor activity. It is likely that the result obtained with the combination of 5′- and 3′-half-molecules is due to the absence of C-A dinucleotide in the anticodon loop.

Identification of a modified nucleoside located in the first position of the anticodon of Torulopsis utilis tRNAPro as 5-carbamoylmethyluridine.

An unknown nucleoside in the first position of the anticodon of Torulopsis utilis tRNAPro has been isolated. The UV, 1H NMR and secondary ion mass spectra indicated that this nucleoside is a uridine derivative, 5-carbamoylmethyluridine. The structure was completely established by comparison of the instrumental analysis results and chromatographic behavior of the isolated nucleoside with those of a synthetic sample.