Yeast Suppressor tRNA Genes

Abstract

The advent of rapid techniques for isolating and characterizing specific eukaryotic DNA sequences offers the promise of studying the structure and function of eukaryotic tRNA genes at the level of individual nucleotides. tRNA genes in any eukaryotic organism are readily accessible by these techniques; both their small size and the availability of purified tRNAs as RNA-DNA hybridization probes facilitate applications of molecular cloning and DNA sequencing to these genes. It has been our view, however, that the complex series of steps by which the coding sequence of a tRNA gene is expressed as a mature, functional tRNA molecule is unlikely to be decipherable by structural studies alone. We have chosen, therefore, to focus our efforts on the molecular cloning and physical analysis of a small set of yeast tRNA genes that have been genetically identified as nonsense suppressors (Hawthorne and Leupold 1974). Twelve genetically mapped loci in yeast correspond to nonsense suppressors of known amino-acid-insertion specificity (Table 1). In only two cases, SUP5 UAG and SUPRL1 UAG (Piper et al. 1976; Piper 1978), have these nonsense suppressor mutations been shown to affect the primary structure of a tRNA, but there is now little doubt that all the loci in Table 1 correspond to tRNA genes. The tyrosine-inserting loci have been particularly intensively studied at the DNA level and a SUP4 UAA gene was shown to be mutant in the DNA sequence specifying a tRNA Tyr anticodon (Goodman et al. 1977; M. V. Olson et al. 1977, 1979, in prep.). HYBRIDIZATION TO RESTRICTION DIGESTS We...