Nucleic Acids Research, 1992, Vol. 20, No. 22 5911-5918
© 1992
MOLECULAR BIOLOGY |
Pseudouridine in the anticodon G
A of plant cytoplasmic tRNATyr is required for UAG and UAA suppression in the TMV-specific context
Institut f
r Biochemie, Bayerische Julius-Maximilians-Universitat, Biozentrum Am Hubland, D-8700 Wrzburg, Germany
* To whom correspondence should be addressed
Received August 7, 1992. Revised October 20, 1992. Accepted October 20, 1992.
We have previously isolated and sequenced Nicotiana cytoplasmic tRNATyr with G*A anticodon which promotes readthrough over the leaky UAG termination codon at the end of the 126 K cistron of tobacco mosaic virus RNA and we have demonstrated that tRNATyr with CM A anticodon is no UAG suppressor. Here we show that the nucleotide in the middle of the anticodon (i.e., t35) also contributes to the suppressor efficiency displayed by cytoplasmic tRNATyr. A tRNATyr with GUA anticodon was synthesized in vitro using T7 RNA polymerase transcription. This tRNATyr was unable to suppress the UAG codon, indicating that nucleotide modifications in the anticodon of tRNATyr have either stimulating (i.e., 
35) or inhibitory (i.e., Q34) effects on suppressor activity. Furthermore, we have shown that the UAA but not the UGA stop codon is also efficiently recognized by tobacco tRNAjA> if placed in the TMV context. Hence this is the first naturally occuring tRNA for which UAA suppressor activity has been demonstrated. In order to study the influence of neighbouring nucleotides on the readthrough capacity of tRNATyr, we have established a system, in which part of the sequence around the leaky UAG codon of TMV RNA was inserted into a zein pseudogene which naturally harbours an UAG codon in the middle of the gene. The construct was cloned into the vector pSP65 and in vitro transcripts, generated by SP6 RNA polymerase, were translated in a wheat germ extract depleted of endogenous mRNAs and tRNAs. A number of mutations in the codons flanking the UAG were introduced by site-directed mutagenesis. It was found that changes at specific positions of the two downstream codons completely abolished the readthrough over the UAG by Nicotiana tRNATyr, indicating that this tRNA needs a very specific codon context for its suppressor activity.
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