Yeast tRNA^Trpgenes with anticodons corresponding to UAA and UGA nonsense codons

Abstract

Naturally occurring suppressor mutants derived from tRNATrp genes have never been identified in S. cerevisiae. Oligonucleotide-directed mutagenesis was used to generate potential ochre and opal suppressors from a cloned tRNATrp gene. In vitro transcription analyses show the ochre suppressor form of the gene, TRPO, accumulates precursors and tRNA in amounts comparable to the parent. The opal suppressor, TRPOP, accumulates 4-5 fold less tRNA. Both forms of the gene are processed and spliced in vitro to produce tRNAs with the expected base sequences. The altered genes were subcloned into yeast vectors and introduced into yeast strains carrying a variety of amber, ochre, and opal mutations. When introduced on a CEN vector, neither ochre nor opal suppressor forms show suppressor activity. Deletion of the CEN region from the clones increases the copy number to 10-20/cell. The opal suppressor form shows moderate suppressor activity when the gene is introduced on this vector, however, the ochre suppressor form exhibits no detectable biological activity regardless of gene copy number. Northern blot analyses of the steady state levels of tRNATrp in cells containing the high copy-number clones reveal 20-100% increases in the abundance of tRNATrp.

Cloning and characterization of two K+ transporters of Debaryomyces hansenii

“…Although this could not be completely discarded since we did not sequence the cDNA or mRNA, this is a highly improbable situation since firstly no sequences corresponding to putative introns present in the genomic cloned sequence were predicted, and secondly there is a high level of conservation between the region where the stop codon was found and the same region of the DoHAK1 sequence obtained from a cDNA. Another possibility was that D. hansenii could have a suppression mechanism, as has been described for several viral and cellular systems including S. cerevisiae (Kim et al, 1990;Bonetti et al, 1995) and C. albicans (Santos et al, 1993). This mechanism could lead to a non-standard translation process of termination codon readthrough in which the stop codon UGA, often described as a particularly leaky codon (Surguchov, 1988), would be avoided.…”

Functional differences among the sixSaccharomyces cerevisiae tRNATrp genes

“…These simple numerical correlations have led to the generalizations that all tRNA genes are functionally equivalent and the relative abundance of tRNA is optimized for maximizing the rate of translation, especially with abundant mRNAs. We and others have shown that misreading of nonsense codons can be influenced by the copy number of genes for tRNA Gln and tRNA Trp (Weiss and Freidberg, 1986;Kim et al, 1990). This implies that fidelity, as well as rate, of translation depends on tRNA abundance.…”

The use of a synthetic tRNA gene as a novel approach to studyin vivotranscription and chromatin structure in yeast

“…tDNAArg(AGG) was obtained from pBR322 clone H13 [19] and subcloned as a 700 bp HhaIlBglll fragment into M13mplO. tDNATrPoP [20] and tDNALeu3 [13] are described in the references. For cloning tDNASYn2, the separate oligonucleotide strands were annealed and ligated into appropriate vectors.…”