Identification of a selenocysteine-specific aminoacyl transfer RNA from rat liver.
The aminoacylation of rat liver tRNA with selenocysteine was studied in tissue slices and in a cell-free system with [75Se]selenocysteine and [75Se]selenite as substrates. [75Se]Selenocysteyl tRNA was isolated via phenol extraction, 1 M NaCl extraction and chromatography on DEAE-cellulose. [75Se]Selenocysteyl tRNA was purified on columns of DEAE-Sephacel, benzoylated DEAE-cellulose and Sepharose 4B. In a dual-label aminoacylation with [35S]cysteine, the most highly purified 75Se-fractions were greater than 100-fold purified relative to 35S. These fractions contained less than 0.7% of the [35S]cysteine originally present in the total tRNA. When [35Se]selenocysteyl tRNA was purified from a mixture of 14C-labeled amino acids, over 97% of the [14C]aminoacyl tRNA was removed. The [75Se]selenocysteine was associated with the tRNA via an aminoacyl linkage. Criteria used for identification included alkaline hydrolysis and recovery of [75Se]selenocysteine, reaction with hydroxylamine and recovery of [75Se]selenocysteyl hydroxamic acid and release of 75Se by ribonuclease. The specificity of [75Se]selenocysteine aminoacylation was demonstrated by resistance to competition by a 125-fold molar excess of either unlabeled cysteine or a mixture of the other 19 amino acids in the cell-free selenocysteine aminoacylation system.
D000818:Animals; D002250:Carbon Radioisotopes; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D008297:Male; D012345:RNA, Transfer, Amino Acid-Specific; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000302:isolation & purification*; D011868:Radioisotopes; D051381:Rats; D011919:Rats, Inbred Strains; D012643:Selenium; D013462:Sulfur Radioisotopes
D002250:Carbon Radioisotopes; D012345:RNA, Transfer, Amino Acid-Specific; D012346:RNA, Transfer, Amino Acyl; D011868:Radioisotopes; D013462:Sulfur Radioisotopes; C036928:tRNA, selenocysteine-; D012643:Selenium