Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNA^Cys

Abstract

In this paper we describe the construction of a yeast tRNACys UGA suppressor. After specific hydrolysis of the parent molecule, the first base of the anticodon GCA was replaced by a uracil. The resulting molecule, harboring a UCA anticodon, was injected into Xenopus laevis oocytes in order to test its biological activities. The level of aminoacylation was similar to that of the parent molecule. Readthrough of the UGA termination codon in /?-globin mRNA, coinjected with the tRNA, indicated suppressor activity; however, tRNACys (anticodon UCA) was a much less efficient suppressor than others tested under the same conditions. We see no post-transcriptional modification of the uracil in the anticodon wobble position after injection into oocytes. This may be related to the low suppressor activity; however, it is also possible that other features of tRNACyS structure may be unadapted to efficient UCA anticodon function.Several lines of evidence now suggest that the overall functional efficiency of a tRNA molecule in protein synthesis is dependent on the structure of the whole of the molecule and its relation to the anticodon. Thus Gorini, observing the varying efficiencies of different nonsense suppressors remarked [I] "it appears that a 'correct' anticodon obtained by mutation can be out of context in the resulting tRNA suppressor molecule". In addition to the occurrence of invariant nucleotides in the tRNA molecule, it is known that base usage in other positions is often far from random [2]. One of the most versatile ways of investigating the importance of particular bases or regions of the molecule is to construct tRNAs with modified primary structure. This may be done either by modification of the DNA or the RNA sequence. Temple et al. have recently reported the construction of an active suppressor tRNALy" by site-directed mutagenesis of the gene [3]. By modification of the RNA sequence, Bruce et al. have succeeded in synthesising a biologically active amber suppressor by modification at the RNA level of yeast tRNAPhe [4]. Here we describe the application of the RNA mutagenic technology to the construction of a potential UGA suppressor with anticodon UCA, by base substitution in yeast tRNACyS. This methodology, dependent largely on the properties of RNA ligase and polynucleotide kinase from phage T4 [5, 61, was initially developed by the group of Uhlenbeck, and applied successfully to the modification of several yeast tRNAs: tRNAPhe [4], tRNAASp [8] and tRNAkg [9].Essentially three reasons led us to select yeast tRNACy" as an interesting species for modification. Among the anticodon changes that may be easily introduced, one should lead to a UGA suppressor species, the activity of which may be readily assayed in vivo as well as in vitro, The normal tRNACYs anticodon, GCA, is conveniently split by ribonuclease TI. Finally, the availability of high specific activity [35S]cysteine should facilitate characterisation of polypeptides synthesized in vivo or in vitro.Enzymes. RNAse TI (EC 3.1.27.3); nuclease S1 (EC 3.1.30...

Yeast tRNATrpgenes with anticodons corresponding to UAA and UGA nonsense codons

“…Highly efficient suppressors can impair cell growth and could be lethal (9). Mutations in the anticodon or adjacent regions may impair the transcription, maturation, or aminoacylation of tRNA (10,1 1) or cause changes in translation context effects rendering them unable to function as a suppressor (12)(13)(14)(15). Very weak suppressors may not be detectable unless the gene is present in multiple copies (16).…”

“…Yeast tRNAPhe and tRNAcYs molecules whose anticodons were altered in vitro are capable of acting as suppressors (12,13,17). To determine whether the single form of yeast tRNATrP could act as a suppressor, the CCA anticodon of a cloned tRNATrp gene was converted, using site-directed mutagenesis, to anticodons corresponding to all three nonsense codons.…”

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs

“…Cys-tRNAs from yeast and plants have also been used to incorporate Cys residues at opal (UGA) codons in vitro and in Xenopus oocytes (Vacher et al 1984;Urban and Beier 1994). To establish tRNA Cys as a viable platform for developing suppressor tRNAs we first characterized E. coli, yeast, and human tRNA Cys (Lipman and Hou 1998) for their aminoacylation efficiency, aa-tRNA stability, and ability to incorporate a modified cysteine residue in two eukaryotic cell-free translation systems, wheat germ (WG) extract, and RRL.…”