RECOVERY OF TRANSFER RNA FUNCTIONS BY COMBINING FRAGMENTED ESCHERICHIA COLI FORMYLMETHIONINE TRANSFER RNA

1. Limited digestion of Escherichia coli tRNAfMet by ribonuclease T1 resulted mainly in a split at -G-D(dihydrouridine)-sequence in the D loop of the tRNA. The resulting 5′- and 3′-half molecules restored the methionine-acceptor activity and the transformylation ability only when mixed. No activities were recovered with either half alone. It was not necessary to preincubate the mixture of the two halves in any particular conditions for the restoration of the activities. 2. The saturation experiment showed that the active complex molecule was formed by a mutual one-to-one interaction of the 5′- and 3′-half molecules. Thermal denaturation experiment revealed that the 5′- and 3′-halves of the tRNA, when mixed, actually interacted to form a complex. However, another 3′-half molecule in which a CAACCA-OH sequence is missing from the 3′-end could not interact to form an active complex. 3. As for the code recognition ability, the reconstituted molecule showed abnormal binding to poly (U4, G) in the absence of ribosome. However, it maintained the ability to recognize the initiator triplets, ApUpG and GpUpG, in the presence of ribosome. As a conclusion, the intactness of the -G-D- sequence in the D loop is not required for the recognition of methionyl-RNA synthetase but is required for the formation of the correct ribosome-mRNA-tRNA complexes. 4. The data were discussed in reference to a possibility that the aminoacyl RNA synthetase recognizes the specificity of tertiary conformation of tRNA molecule rather than the specificity of primary nucleotide sequences. An important participation of the nucleotide sequence at the 3′-end of the tRNA molecule was also discussed on the basis of the present data. © 1969.