Pseudouridine in the anticodon GΨA of plant cytoplasmic tRNA^Tyris required for UAG and UAA suppression in the TMV-specific context

Abstract

We have previously isolated and sequenced Nicotiana cytoplasmic tRNA(Tyr) with G psi 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 tRNA(Tyr) with Q psi A anticodon is no UAG suppressor. Here we show that the nucleotide in the middle of the anticodon (i.e., psi 35) also contributes to the suppressor efficiency displayed by cytoplasmic tRNA(Tyr). A tRNA(Tyr) with GUA anticodon was synthesized in vitro using T7 RNA polymerase transcription. This tRNA(Tyr) was unable to suppress the UAG codon, indicating that nucleotide modifications in the anticodon of tRNA(Tyr) have either stimulating (i.e., psi 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 tRNA(G psi ATyr), if placed in the TMV context. Hence this is the first naturally occurring tRNA for which UAA suppressor activity has been demonstrated. In order to study the influence of neighbouring nucleotides on the readthrough capacity of tRNA(Tyr), 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 tRNA(Tyr), indicating that this tRNA needs a very specific codon context for its suppressor activity.

Lack of Pseudouridine 38/39 in the Anticodon Arm of Yeast Cytoplasmic tRNA Decreases in Vivo Recoding Efficiency

“…We did not obtain an effect on the efficiency of stop codon readthrough with the yeast pus1⌬ strain, despite the fact that the three natural yeast suppressors of UAG in the TMV context are all substrates of Pus1p. The presence of ⌿35 in tRNA G⌿A Tyr was shown to be necessary for efficient suppression in vitro of the UAG of TMV RNA (29), probably because it stabilizes the codon-anticodon interaction (56). However, formation of ⌿35 in tRNA G⌿A Tyr remains unaffected in a pus1⌬ strain, although recombinant Pus1p can catalyze in vitro the formation of ⌿35 in a transcript of intron-containing tRNA G⌿A Tyr (14).…”

“…Changes in tRNA structure, such as those induced by defective modification, may affect the decoding properties of tRNA. For example, lack of ⌿35 in yeast and plant tRNA G⌿A Tyr (28,29) and ⌿38/39/40 in several E. coli tRNAs (Ref. 30 and reviewed in Ref.…”

An extended structural signature for the tRNA anticodon loop

“…As deduced from the tRNA sequences extracted from the tRNA database (Sprinzl et al+, 1998), modified nucleotides are absent at position 33, and are very rare at position 35 (,1%) and 36 (,2%) (Auffinger & Westhof, 1998b)+ At the latter locations, modifications of the Watson-Crick sites would prevent recognition of the mRNA+ Similarly, at position 35, methylation of the (R)N7 and the (Y)C5 sites would prevent the formation of the identified interaction between the base of residue 35 and the ribose of residue 33+ Interestingly, the only modified base that is tolerated at position 35 is a pseudouridine (⌿) in which the less hydrophilic C5-H group found in pyrimidines is replaced by a N1-H group (Charette & Gray, 2000)+ This observation constitutes a strong evidence for the formation of a (U33)O29 + + + H-C5(Y35) interaction because a ⌿35 allows for the formation of a (U33)O29 + + + H-N1(⌿35) hydrogen bond in replacement of the weaker (U33)O29 + + + H-C5(U35) interaction without perturbing the loop structure (Fig+ 4)+ On the basis of experimental data, it has been proposed that the ⌿35 modification increases the activity of yeast suppressor tRNA Tyr (U⌿A) by increasing the stability of the anticodon(⌿)-codon(A) interaction (Johnson & Abelson, 1983)+ In plants, modified cytoplasmic tRNA Tyr (G⌿A) is required for an efficient recognition of the UAG or UAA codons, whereas the unmodified GUA anticodon does not recognize the same UAG or UAA codons (Zerfass & Beier, 1992)+ Recently, it has been proposed that ⌿35 in echinoderm mt tRNA Asn is involved in the decoding of the three AAC, AAU, and AAA codons, whereas the U35 variant can only decode the two AAC and AAU codons (Tomita et al+, 1999)+ In these systems, strengthening of the anticodon(⌿35)-codon(A) interaction is necessary and is helped presumably through the formation of a (⌿35)N1-H + + + O29(U33) hydrogen bond equivalent to a (U35)C5-H + + + O29(U33) interaction+ Several biochemical studies with natural or chemically modified nucleotides at various locations of the anticodon loop were designed with the goal of assessing the existence and significance of specific tertiary interactions+ It has been shown that modifications of U33 do not affect the aminoacylation ability of yeast tRNA Phe (Wittenberg & Uhlenbeck, 1985) for which a canonical structure of the anticodon loop is not required+ Yet, numerous examples indicate that modifications of U33 (for example, U33 to m 3 U, C33, dU33, Um, m 6 U, or D) introduced in wild-type tRNA sequences (Uhlenbeck et al+, 1982;Dix et al+, 1986) and tRNA transcripts (von Ahsen et al+, 1997; Ashraf et al+, 1999aAshraf et al+, , 1999b alter the translational efficiency, supporting the idea that the ability of tRNA anticodon hairpins to adopt a canonical structure involving an intact U-turn is mandatory for ribosome binding+ Methylation of the N3 atom that prevents the formation of the (U33)N3-H + + + OR-P(A36) interaction shows especially strong effects on the translational efficiency (Uhlenbeck et al+, 1982;Dix et al+, 1986; von Ahsen et al+, 1997)+ Similarly, methylation of the U33 ribose of yeast tRNA Phe...…”

Partial Purification and Characterization of Nuclear Ribonuclease P from Wheat

“…The standard substrate used in processing reactions corresponds to nuclear pre-tRNATYr from Nicotiana rustica, containing the five-nucleotide natural S f l a n k (preceded by an additional G to facilitate transcription by T7 polymerase) and a mature 3'-CCA end, but lacking the intron. The gene encoding this pre-tRNA had been put under control of the T7 promoter and cloned into pUC19 to obtain pNtYdT7 (Zerfass and Beier, 1992).…”