Makoto Kawakami

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].

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 .

Protein assays in very dilute solutions

Abstract The protein content determination in very dilute solutions was accomplished by the combination of two methods. In the first one the proteins were coprecipitated and in the second they were radiolabeled.

The gene coding for lipoprotein signal peptidase (lspA) and that for isoleucyl-tRNA synthetase (ileS) constitute a cotranscriptional unit in Escherichia coli

The lspA gene coding for lipoprotein signal peptidase is located very close to the ileS gene coding for isoleucyl‐tRNA synthetase on the Escherichia coli chromosome. Deletions were generated in vitro from both ends of the 4.3 kb fragment that carries the lspA gene and the ileS gene, and the expression of the two genes was examined before and after insertion of the trp promoter‐operator at one end. The results indicate that the lspAandileS genes constitute a cotranscriptional unit in the order of promoter‐ileS‐lspA. The gene order of dnaJ‐rpsT‐ileS‐lspA‐dapB around 0.5 min on the E. coli chromosome map was also determined.

Isolation of gram quantities of isoleucyl‐tRNA synthetase from an overproducing strain of Escherichia coli and its use for purification of cognate tRNA

The ileS gene coding for isoleucyl‐tRNA synthetase was cloned on a runaway‐replication plasmid. From the cells harboring the plasmid, gram quantities of the synthetase were isolated using two column procedures. The synthetase was used for the purification of cognate tRNA. Isoleucine tRNAGAU of greater than 90% purity was easily isolated by taking advantage of a specific complex formation of the synthetase with cognate tRNA.