The oligodeoxyribonucleotide ethyl phosphotriesters d-Tp(Et)Gp(Et)G and d-Tp(Et)Tp(Et)Cp(Et)A, which are complementary to the 3′-CpCpA terminus and -UpGpApA- anticodon region, respectively, of tRNAphe coli have been used as in vitro probes of the structure and function of tRNA. The effect of these triesters on the aminoacyl-tRNA synthetase catalyzed aminoacylation of tRNA was examined. At 0° both triesters inhibit the formation of phenylalanyl-tRNAphe by approximately 50-60%. The inhibition decreases with increasing temperature. A Lineweaver-Burk analysis at 0° shows that the inhibition by both triesters is competitive in nature. The results suggest that the inhibition is a consequence of the formation of complexes between the triesters and the tRNA as described in the preceding paper (Miller, P. S., Barrett, J. C., and Ts'o, P. O. P. (1974), Biochemistry 13, 4887). As expected, d-Tp(Et)Gp(Et)G inhibits the aminoacylation of all five tRNAs examined (phenylalanine, lysine, tyrosine, proline, and leucine) by 55-80%, while the inhibition by d-Tp(Et)Tp(Et)Cp(Et)A is more specific and is greatest for tRNAphe. The effect of the triesters on various related reactions catalyzed by the aminoacyl-tRNA synthetases was investigated. d-Tp(Et)Gp(Et)G inhibits the enzymic deacylation of PhetRNAphe at 0° by 15%, but has no effect on the synthetasecatalyzed ATP-pyrophosphate exchange reaction, d-Tp(Et)Tp(Et)Cp(Et)A does inhibit pyrophosphate exchange; however, the inhibition is not specific for any amino acid. The transfer of an activated amino acid from a preformed aminoacyl adenylate-synthetase complex to tRNA at 0° is inhibited by d-Tp(Et)Gp(Et)G for three amino acids (phenylalanine, tyrosine, and leucine), while d-Tp(Et)Tp(Et)Cp(Et)A only inhibits the transfer of phenylalanine. The effect of d-TpGpG, d-GpGpT, d-TpTpCpA, UpGpG, GpGpU, and UpUpCpA on aminoacylation of tRNAphe was examined. At 0°, the inhibitory activities of these oligonucleotides directly parallel the magnitudes of their association constants with tRNAphe as determined by equilibrium dialysis. These findings demonstrate that the triesters can inhibit tRNA aminoacylation by specifically masking complementary regions of the tRNA through complex formation. The results are discussed in terms of the current mechanism of aminoacylation of tRNA and of the role of the anticodon in the tRNA-synthetase interaction.
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