Isolation and Characterization of Amber Suppressors in Yeast

Abstract

Nonsense suppressors were obtained in a haploid yeast strain containing eight nutritional mutations, that are assumed to be amber or ochre, and the cyc1-179 amber mutation that has a UAG codon corresponding to position 9 in iso-1-cytochrome c. Previous studies established that the biosynthesis and function of iso-1-cytochrome c is compatible with replacements at position 9 of amino acids having widely different structures (Stewart and Sherman 1972). UV-induced revertants, selected on media requiring the reversion of one or two of the amber nutritional markers, were presumed to contain a suppressor if there was the unselected reversion of at least one other marker. The 1088 suppressors that were isolated could be divided into 78 phenotypic classes. Only 43 suppressors of three classes caused the production of more than 50% of the normal amount of iso-1-cytochrome c in the cyc1-179 strain. Genetic analyses indicated that all of these highly efficient amber suppressors are allelic to one or another of the eight suppressors which cause the insertion of tyrosine at ochre (UAA) codons (Gilmore, Stewart and Sherman 1971). Furthermore, only tyrosine has been identified at position 9 in iso-1-cytochrome cin cyc1-179 strains suppressed with these efficient amber suppressors.

Inhibition of Growth by Amber Suppressors in Yeast

“…Similarly, excessive UAA suppression appears to cause the lethal effect observed when the efficiency of the tyrosine-inserting UAA suppressors is increased by the ++ modifier ( Cox 1965Cox , 1971LIEBMAN, STEWART and SHERMAN 1975b). Amber (UAG) suppressors which act with approximately a 75% efficiency on the amber mutant cycl-179 have been isolated (SHERMAN et al 1973;LIEBMAN, SHERMAN and STEWART 1976). Here we report that in yeast, unlike in E. coli, highly efficient amber suppression is associated with reduced growth rates.…”

“…Estimation of mutation rate of SUP5-a2: As will be described in detail below, slowly growing strains, which contain the highly efficient suppressor SUP5a2, give rise to fastergrowing strains which have lower efficiencies of suppression. The proportions of the two types of cells can be formulated from the following two equations: SHERMAN and STEWART 1976).…”

“…The efficiencies of yeast nonsense suppressors have been estimated from the level of iso-I -cytochrome c in these suppressed mutants. Such measurements, which were accomplished by spectroscopic examinations of intact cells as well as by quantitative extraction of iso-l-cytochrome c, have indicated that a given suppressor acts on different nonsense mutants with various levels of efficiencies (GILMORE, STEWART and SHERMAN 1971 ; LIEBMAN, SHERMAN and STEWART 1976; LIEBMAN, STEWART and SHERMAN 1975a), even though functional proteins are formed with apparently most or all amino acids at these nonsense sites. However.…”

“…It is evident that a more precise measure of efficiency of suppression can be obtained with cycl mutants than with nutritional mutants since the levels of suppressed iso-I -cytochrome c can be determined directly instead of being inferred from growth rate. In fact, the levels of iso-l-cytochrome c were used to distinguish between different amber suppressors that appeared to act with equal efficiencies on all of the nutritional markers used for testing amber suppressors ( LIEBMAN, SHERMAN and STEWART 1976).…”

Labeling and Single-Molecule Methods To Monitor G Protein-Coupled Receptor Dynamics

“…Their approach, known as amber codon suppression, was inspired by the earlier discoveries of amber suppressors in E. coli and yeast. , Amber suppressors are certain tRNA species that are capable of suppressing the translation termination signal of the amber codon to yield a readthrough polypeptide. In eukaryotic cells, the complex of two polypeptide chain release factors, eRF1 and eRF3, mediates translation termination .…”