Polyamine requirement for efficient translation of amber codons in vivo.

Abstract

Multiplication of several amber mutants of bacteriophage T7 was decreased in two polyamine-deficient mutants of Escherichia coli K-12 carrying amber suppressors, relative to the multiplication ofwild type bacteriophage T7 in the same hosts. In contrast the same T7 amber bacteriophages multiplied well in these strains when supplemented with polyamines. The requirement for polyamines for optimal translation of amber codons in vivo was confirmed by showing that infection of polyamine-depleted E. coli with bacteriophage T7 carrying an amber mutation in gene I resulted in an increased accumulation ofthe amber fragment of the gene 1 protein and a decreased accumulation of the full-length gene 1 protein compared with infection of an aminesupplemented culture. These results indicate that one important function of polyamines in vivo is concerned with protein translation and the protein-synthesizing ribosomal complex.Although many effects ofputrescine and spermidine have been observed in in vitro systems (for references, see refs. 1-3), we still do not know the biological role of these amines in vivo. For this reason we have developed mutants of Escherichia coli that have deletions in the genes for the biosynthetic enzymes for these amines and hence contain no amines when grown in an amine-free medium (4, 5). The organisms that we first described could grow indefinitely in the absence of amines but did so at a lowered growth rate.One of the postulated roles for polyamines is an effect on some aspect of protein biosynthesis, and we decided to see if a role in protein synthesis could be demonstrated in amine-deficient mutants in vivo. In previous experiments (6), we showed that transduction of certain rpsL (strA)* alleles into an aminerequiring strain converted this organism from one with a partial requirement for amines for growth into one with an absolute requirement. Because rpsL mutations are known to cause defects in the S12 ribosomal protein and to affect the fidelity of translation in vitro and in vivo (8-10), our studies suggested that polyamines might act by affecting the stability and conformation of the protein-synthesizing ribosomal complex.The experiments in the present paper examined the effect of polyamines on translation from a different point of view: namely, does polyamine deprivation affect the translation of amber codons in vivo? We have shown that, in E. coli, polyamine deficiency causes a marked decrease in the multiplication of some amber mutants of bacteriophage T7, compared to the multiplication ofwild type bacteriophage T7, even though these bacterial strains contain a suppressor. However, when these strains are grown in the presence of polyamines, they are suitable hosts for the amber mutants of bacteriophage T7 as well as the wild type. These results indicate that polyamines are required for the optimal translation of an amber codon in a strain carrying a suppressor and support the hypothesis that an important function of the polyamines is concerned with the protein-synthesizing ribosom...

Nonsense mutations of the ornithine decarboxylase structural gene of Neurospora crassa.

“…Symbols: 0, growth on minimal medium; 0, growth after addition of arginine; A, growth after addition of ornithine to arginine-treated culture. Polyamines have been shown to influence the fidelity of protein synthesis and to be required in some cases for the action of nonsense suppressor mutations in bacteria (1,11,12,24,25). Our data show that nonsense suppression is qualitatively successful in N. crassa despite severe polyamine starvation in our suppressed mutants.…”

Polyamine Titer in the Embryonic Axis and Cotyledons of Glycine max (L.) during Seed Growth and Maturation

“…Polyamines act in the cells as organic cations at physiological pH. Studies primarily with microorganisms and mammalian cells have yielded supporting evidence for participation of polyamines in protein synthesis (14) and enzyme regulation (6). In higher plants, the gene(s) and/or formation ofa-amylase in isolated barley aleurone layers (8).…”

It All Started on a Streetcar in Boston

“…In related experiments, we later showed that although wild-type bacteriophage T4 grew normally in polyamine-deficient cells, T4 phage containing an amber mutation grew poorly even if the host contained a suppressor tRNA; the latter cells accumulate an amber fragment (71). Therefore, these experiments indicated that the efficiency of nonsense suppression by a suppressor tRNA is very poor in polyamine-deficient cells.…”