Leif A. Isaksson

Translation rates and misreading characteristics of rpsD mutants in Escherichia coli.

SummaryThree ribosomal ambiguity (Ram) mutants, changed in ribosomal protein S4, have been examined with respect to elongation rate and misreading of translation in vivo and in vitro. Ram mutants increase misreading of nonsense codons in vivo, compared to wild type, between 2–50 times depending on the nature of the nonsense codon, its position, and which rpsD allele is present. Ram ribosomes also show an increased error frequency in vitro. The elongation rate of translation does not seem to be significantly changed, neither in vivo nor in vitro, irrespective of which rpsD allele is present.We suggest that there exists no general relationship between the accuracy and the overall speed of translation in Ram strains.

A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for tRNA nonsense suppressors

SummaryA spontaneous mutant was isolated that harbors a weak suppressing activity towards a UAG mutation, together with an inability to grow at 43° C in rich medium. The mutation is shown to be associated with an increased misreading of UAG at certain codon contexts and UAA. UGA, missense or frameshift mutations do not appear to be misread to a similar extent. The mutation gives an increased efficiency to several amber tRNA suppressors with-out increasing their ambiguity towards UAA. The ochre suppressors SuB and Su5 are stimulated in their reading of both UAG and UAA with preference for UAG. An opal suppressor is not affected. The effect of the mutation on the efficiency of amber and ochre suppressors is dependent on the codon context of the nonsense codon.The mutated gene (uar) has been mapped and found to be recessive both with respect to suppressor-enhancing ability as well as for temperature sensitivity. The phenotype is partly suppressed by the ochre suppressor SuC. It is suggested that uar codes for a protein, which is involved in translational termination at UAG and UAA stop codons.

A procedure for isolation of spontaneous mutants with temperature sensitive synthesis of RNA and/or protein

SummaryA procedure for the isolation of spontaneous temperature sensitive mutants of Escherichia coli has been developed. They are selected as survivors at high temperature against the combined killing effects exerted by a temperature inducible lambda prophage and either streptomycin plus ampillicin or ampicillin plus cycloserine. The mutants so obtained are blocked in vivo in the synthesis of RNA or protein or both at restrictive temperature.

Analysis of rpsD mutations in Escherichia coli

SummaryA certain proportion of protein S7 exists in an altered form in E. coli rpsD (S4) mutants. Depending on the type of S4 mutation involved, two different forms of the altered S7 can be distinguished. The unusual form is longer than normal S7 by about 500 daltons due to extra material at the carboxyl end of the protein. It is suggested that a mutationally altered S4 might lower the efficiency of termination during translation of the messenger for S7. This results in an increased frequency of translational read-through, which gives the observed longer forms of S7. Data are interpreted to mean that one class of S4 mutants might suppress UGA and UAG whereas another class only suppresses UGA.

Analysis of rpsD mutations in Escherichia coli. III: Effects of rpsD mutations on expression of some ribosomal protein genes

SummaryRelative rates of production and steady state levels of ribosomal proteins were determined in a temperature sensitive rpsD (S4) mutant of Escherichia coli. Some proteins (S4, S12, S13) were overproduced in the mutant at permissive temperature but steady state levels of all examined ribosomal proteins were normal. In a rpsD+/rpsD+ homodiploid strain the relative rates of production of ribosomal proteins were not affected by the increased gene dose. In a rpsD+/rpsD heterodiploid strain only wild type, but not mutant S4, was found. In such a strain S4, S7, S12 and probably S13 is overproduced. It is implied that S4 is involved in the regulation of expression of proximal genes of the two transcriptional units including the genes coding for S4 itself and S12, respectively. A degradation system for ribosomal proteins, which is rapid enough to be of regulatory significance, is demonstrated.

Studies on microbial RNA. V. A comparison of the in vivo methylated components of ribosomal RNA from Escherichia coli and Saccharomyces cerevisiae.

Abstract 1. Methylated bases in ribosomal RNA (rRNA) were estimated by a method consisting of (a) in vivo methyl labelling of the rRNA, (b) isolation of the rRNA, (c) acid hydrolysis of the RNA in the presence of carriers, (d) separation of the hydrolysis mixture by two-dimensional chromatography and (e) estimation of the radioactivity in the separated spots. Ribose-methylated nucleotides were estimated separately. 2. The procedure was used to compare five strains of Escherichia coli and two strains of Saccharomyces cerevisiae. The two organisms differ markedly in the distribution of methylated compounds in their rRNA, while strains of the same organism are very similar. The content of O- ribose methyl groups was high in the yeast strains but rather low in the bacteria.

Antagonistic effects of mutant elongation factor Tu and ribosomal protein S12 on control of translational accuracy, suppression and cellular growth

Kirromycin-resistant mutant forms of elongation factor Tu, which are coded by tufA (Ar) or tufB (Bo) and are associated with an increased rate of translational error formation, have been analysed. In vivo, Ar was found to increase misreading as well as suppression of non-sense codons irrespective of Bo in a strain with wild type ribosomes. It is therefore not necessary to evoke both tufA (Ar) and tufB (Bo) mutations together in order to increase translational error as suggested earlier [1]. When combined with a hyperaccurate ribosomal rpsL (S12) mutation, Ar counteracts the restrictive effects on translational error formation caused by the altered protein S12, thus restoring the levels of missense error in vitro and non-sense error and suppression in vivo to near wild type values. As judged from in vitro experiments this results principally from a lowered selectivity of the Ar ternary complex at the initial discrimination step on the ribosome during translation. In vivo, this compensatory effect on the rpsL mutation on non-sense error formation and suppression is seen irrespective of the nature of tRNA or codon context. Furthermore, the tufA mutation enhances the cellular growth rate of the rpsL mutant, whereas it decreases growth of strains with normal ribosomes. Inactivation of one of the two genes coding for EF-Tu (tufB), while leaving the other gene (tufA) intact, can by itself, increase non-sense error formation and suppression.

Pleiotropic effects of ribosomal protein S4 studied in Escherichia coli mutants

SummaryA temperature sensitive mutant of Escherichia coli was found to have two mutational alterations of its ribosomes: one of these was a streptomycin dependent mutation and the other was a suppressor alteration of S4, with a marked structural change. The altered form of S4 was studied in a strain that was constructed so that this alteration was the only one effecting the structure of the ribosome. Here, it was shown that the mutant form of S4 cause a temperature sensitive defect in the assembly of 30S subunits in vivo which is reflected in the inability of this mutant to properly process ribosomal RNA at the restrictive temperatures. An analysis of both transductants and revertants of this mutant show that the suppression of the streptomycin dependence phenotype, temperature sensitivity, and a defect in RNA processing all have their origin in a single mutational event effecting the structural gene for S4.

Aminoacylation and polypeptide synthesis with tRNA lacking ribothymidine

Abstract The role of thymine in tRNA has been investigated in vitro , using a mutant of Escherichia coli completely lacking thymine in its tRNA. In both aminoacylation and polypeptide synthesis, the thymine-free tRNA seems to behave identically with normal tRNA.

Temperature-sensitive mutants in cysteinyl-tRNA ligase of E. coli K-12

SummaryTwo mutants with a defective cysteinyl-tRNA synthetase have been found in a collection of spontaneous temperature sensitive mutants. The mutated gene, which is designated cysS, is closely contransduced with purE.