Replacement and insertion of nucleotides at the anticodon loop of E. coli tRNAMetf by ligation of chemically synthesized ribooligonucleotides.
Insertion of the four major nucleotides at the 5'-side of the anticodon triplet of E. coli tRNAMetf was performed by joining of the half molecules obtained by 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.
D000926:Anticodon / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis / Q000138:chemical synthesis*; D012270:Ribosomes / Q000378:metabolism
D000926:Anticodon; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D012346:RNA, Transfer, Amino Acyl; C026464:tRNA(m)(Met), methionine-; D012343:RNA, Transfer