Nucleic Acids Research, 1985, Vol. 13, No. 10 3685-3697
© 1985
Articles |
Replacement and Insertion of nucleotides at the anticodon loop of E. coli tRNAfMet by ligation of chemically synthesized ribooligonucleotides
Faculty of Pharmaceutical Sciences, Osaka University Suita, Osaka 565 *Faculty of Pharmaceutical Sciences, Hokkaido University Sapporo 160, Japan
Received March 4, 1985. Revised April 30, 1985. Accepted April 30, 1985.
Insertion of the four major nucleotides at the 5'-side of the anticodon triplet of E. coli tRNAMet was performed by joining of the half molecules obtained b limited digestion with RNase A and the chemically synthesized tetranucleotide pN-C-A-U using RNA ligase. Insertion of U-U at the 5'-side or A and A-A at the 3'-side of the anticodon were also performed using U-U-C-A-U, C-A-U-A and C-A-U-A-A. The constant U next to the 5'-side of the anticodon was replaced with A and C by ligation of A-C-A-U and C-C-A-U to the 5'-half molecule which had been treated with periodate plus lysine, followed by joining to the 3'-half. These modified tRNAs were tested for their ability to accept methionine with the methionyl-tRNA synthetase of E. coli The affinity of these analogs for the synthetase decreased more extensively when the insertion was at the 3'-side of the anticodon triplet. Insertion of mononucleotides at the 5'-side or replacement of the constant U next to the 5';-side of the anticodon did not affect aminoacylation drastically. This may mean that the 3'-side of the anticodon loop of tRNA is one of the major recognition sites for the methionyl-tRNA synthetase.