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Nucleic Acids Research, 1983, Vol. 11, No. 12 3863-3872
© 1983

MOLECULAR BIOLOGY

Modification of the anticodon triplet of E.coli tRNAfMet by replacement with trimers complementary to non-sense codons UAG and UAA

Eiko Ohtsuka, Takefumi Doi, Ryoichi Fukumoto, Jitsuhiro Matsugi and Morio Ikehara

Faculty of Pharmaceutical Sciences, Osaka University 1–6 Yamadaoka, Osaka 565, Japan

Received April 26, 1983. Revised May 24, 1983. Accepted May 24, 1983.

E. coli tRNAfMet was hydrolyzed with RNase A using a limited amount of the enzyme to give two half molecules lacking the anticodon trimer and 3'-terminal dimer. Chemically synthesized trimers CUAp and UUAp were joined to the 5'-half molecules by phosphorylation with polynucleotide kinase plus ATP followed by treatment with RNA ligase. These modified tRNAfMet, species had anticodons complementary to the termination codons UAG and UAA. Two half fragments were joined by a similar procedure to yield a molecule lacking the anticodon trimer and the 3'-dimer. Methionine acceptor activity of these tRNA was tested under conditions in which the CAU inserted control tRNAfMet, accepted methionine. It was found that all three modified molecules were not recognized by the methionyl-tRNA synthetase from E.coli. The other sixteen amino acids were not incorporated with partially purified aminoacyl-tRNA synthetases.


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