Biopolymers and cell. 2006; 22 (3): 201 - 209

The isolation of histidine tRNA from Thermus thermophilus and the study of its primary structure and interaction sites with homologous aminoacyl-tRNA synthetase

O. I. Gudzera, I. A. Krikliviy, A. D. Yaremchuk, M. A. Tukalo

Histidine tRNAs (tRNA^His) are unique in possing an extra 5´-base (G-1) not found in other tRNAs. To study the molecular mechanisms of tRNA^His interaction with histidyl-tRNA synthetase, the method for purification of tRNA^His from Thermus thermophilus has been developed and tRNA1^His has been sequenced. tRNA^His from Thermus thermophilus was isolated by combination of low-pressure benzoyl-DEAE- cellulose and DEAE Toyopearl 650 chromatographies with HPLC on DEAE 5PW and reversed phase columns. The nucleotide sequence of T.thermophilus tRNA1^His has been determined by rapid gel-sequencing method. tRNA1^His from T.thermophilus is different from those of E.coli at 23 positions. The sites of interaction of tRNA^His with histidyl-tRNA synthetase have been studied by the method of chemical modification with ethylnitrosourea. Histidyl-tRNA synthetase protects from modification following phospates: 8 – between acceptor and D-stems, 27, 28, 29 from 5´-end of anticodon stem, 34 – at the anticodon and phosphates 67, 68 from 3´-end of acceptor stem. All the protected sites of tRNA^His are found on one side of three-dimensional structure of tRNA where the variable stem is also located. D-stem is located on the opposite side and does not interact with the enzyme.

Keywords: tRNA, aminoacyl-tRNA synthetase, RNA-protein recognition, chemical modification.