Nucleotide sequences and functional characterization of two tobacco UAG suppressor tRNA(Gln) isoacceptors and their genes

We isolated and sequenced the two major tRNA(Gln) isoacceptors with CUG and UmUG anticodons from the cytoplasm of Nicotiana rustica. These are the fir st tRNAs(Gln) of nuclear origin characterized in plants. The tRNA(Gln) sequ ences were used to design probes for the isolation of the corresponding gen es from a nuclear DNA library of N. rustica. The two cloned Nicotiana tRNA( Gln) genes, coding for either of the two isoacceptors, are efficiently tran scribed in HeLa cell nuclear extract. In vitro translation in the presence of purified Nicotiana tRNAs(Gln) was carried out in a wheat germ extract pa rtially depleted of endogenous tRNAs. Cytoplasmic (cyt) tRNA(CUG)(Gln) and to a lesser extent cyt tRNA(UmUG)(Gln) Stimulated readthrough over the UAG stop codon present in the tobacco mosaic virus-specific context. The two tR NAGln isoacceptors are the second class of natural UAG suppressors identifi ed in plants, in addition to cyt tRNA(G Psi A)(Tyr) which has previously be en characterized as the first natural UAG suppressor.