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Construction, aminoacylation and 80 S ribosomal complex formation with a yeast initiator tRNA having an arginine CCU anticodon
Author(s) -
Beauchemin Nicole,
Grosjean Henri,
Cedergren Robert
Publication year - 1986
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(86)80639-1
Subject(s) - transfer rna , aminoacylation , biochemistry , ribosomal rna , biology , methionine , yeast , recombinant dna , microbiology and biotechnology , chemistry , rna , amino acid , gene
The digestion of yeast initiator methionine tRNA with mung bean nuclease and U 2 ribonuclease yielded 5'‐ and 3'‐fragments, respectively. These two fragments together represent the entire tRNA sequence except for A 35 , the central nucleotide of the anticodon, and the CCA terminus. Using RNA ligase, a cytosine was added and the anticodon loop having a C 35 was reformed. Subsequent treatment of this product with CCA‐transferase yielded a full‐length methionine tRNA having an arginine CCU anticodon. This recombinant tRNA Met (CCU) was charged with methionine by the yeast tRNA synthetase. Aminoacylation of the recombinant was however less extensive than in the case of native tRNA Met (CAU). After aminoacylation the recombinant tRNA formed an 80 S ribosomal complex.