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L A Isaksson

Publications and source records attributed to L A Isaksson.

At least 19 recordsLinked to original sources
PubMed1993

Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20.

A spontaneous mutant of Escherichia coli K-12 was isolated that shows an increased misreading ability of all three nonsense codons together with an inability to grow at 42 degrees C. It is demonstrated that the mutation is a deletion of the gene rpsT, coding for ribosomal protein S20. The loss of this protein not only influences the decoding properties of the ribosome; the modification pattern of 16S ribosomal RNA is also changed. This leads to a deficiency in the ability of the mutant to associate its 30S subunits with 50S subunits to form 70S ribosomes. It is suggested that two modified bases, m5C and m6(2)A, are directly or indirectly essential for association of subunits to functional ribosomes in the rpsT mutant strain. Two other modifications were also studied; m2G which is not affected at all and m3U which is undermodified in both active and inactive subunits and, therefore, not involved in subunit association.

Bacterial Proteins↗
PubMed1991

Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo.

The binding of the EF-Tu.GTP.aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23S ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23S rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as normal or mutant ribosomal protein S12 (rpsL). Together with wild-type EF-Tu, the 2661C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wild-type form). Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2661C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa). The lethal effect obtained by the combination of 2661C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain. In such a viable strain, 2661C had no effect on either the translation elongation rate or nonsense codon suppression. Our data suggest that the G base at position 2661 in 23S rRNA is important for binding of EF-Tu during protein synthesis in vivo. The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.

Alleles↗
PubMed1991

Codon choice and potential complementarity between mRNA downstream of the initiation codon and bases 1471-1480 in 16S ribosomal RNA affects expression of glnS.

A cis-acting expression mutation, GAG to GAA, in the third codon of the glnS gene is analyzed. Both codons code for glutamic acid but the mutation is known to increase gene expression by four fold. We show that the mutation has an effect only if it is located in the beginning of a gene but not if located internally. Data are presented that suggest that the reason for the increased expression by the mutation is the potential formation of one more base pair between the mRNA and 16S ribosomal RNA. Gene expression varies about 16 fold as the number of potential base pairs within the sequence 1471-1480 in 16S RNA increase from two to ten. We also give evidence that supports the idea that the presence of rare codons near the beginning of the mRNA can affect expression.

Arginine↗
PubMed1990

Antisuppression by mutations in elongation factor Tu.

Two slow-growing kirromycin-resistant Escherichia coli mutants with altered EF-Tu (Ap and Aa) were studied in vivo in strains with an inactive tufB gene. Mutant form Aa was isolated as an antisuppressor of the tyrT(Su3) nonsense suppressor, as described here. Ap, the tufA gene product of strain D2216 (from A. Parmeggiani), has previously been shown to give an increased GTPase activity. The slow cellular growth rates of both EF-Tu mutants are correlated with decreased translational elongation rates. Ap and Aa significantly decrease suppression levels of both nonsense and missense suppressor tRNAs [tyrT(Su3), trpT(Su9), glyT(SuAGA/G)], but have only little or no effect on misreading by wild-type tRNAs. A particular missense suppressor, lysT(SuAAA/G), which acts by virtue of partial mischarging as the result of an alteration in the amino acid stem, is not significantly affected by the EF-Tu mutations. The combination of tufA(Aa) and a rpsD12 ribosomal mutation is lethal at room temperature and the double-mutant strain has an elevated temperature optimum (42 degrees C) for growth rate, translation rate and nonsense suppression. Our data indicate an alterated interaction between Aa and the ribosome, consistent with our in vitro results.

Amino Acids↗
PubMed1988

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.

Codon↗
PubMed1988

Contribution of normal and error-prone ribosomes to translational error formation in vivo.

Introduction of tRNA missense suppressors, and/or a protease deficiency into Escherichia coli strains has no significant effect on misreading of non-sense codons. An increased cellular level of faulty proteins therefore does not seem to have much secondary effect on translational accuracy. A genetic test system with two UGA non-sense mutations in the same fused lacIlacZ gene does not demonstrate any enrichment of error-prone ribosomes after read-through of the first non-sense codon in such strains. In contrast, the addition of sublethal amounts of streptomycin to a wild type strain appears to enrich error-prone ribosomes at the second non-sense codon, indicating the existence of a subpopulation of streptomycin-binding ribosomes. Ribosomes in a ribosomal ambiguity mutant strain (rpsD) with or without tRNA missense suppressors appear to be functionally homogeneous with respect to error production, as judged by read-through of the double UGA codons. The results that the major contribution to translational error formation in vivo originates from normal ribosomes and not from error-prone defective particles. An increased translational error in a bacterium results in very little, if any, increased functional heterogeneity of the ribosomal population with respect to error production. This suggests that an autocatalytic formation of translational errors is unlikely to occur in a growing bacterium.

Codon↗
PubMed1988

Is efficiency of suppressor tRNAs controlled at the level of ribosomal proofreading in vivo?

Ribosomal rpsD mutations did not stimulate nonsense suppressor tRNAs in a general manner according to their increased ribosomal ambiguity and decreased proofreading efficiency. Streptomycin, which stimulates error production by blocking proofreading in vitro, did not increase efficiency of suppressor tRNAs in strains with normal or streptomycin-resistant (rpsL) ribosomes. It did so only in combination with one rpsL mutation which is associated with streptomycin pseudodependence.

Codon↗
PubMed1986

Primary structures of mutationally altered ribosomal protein L7/L12 and their effects on cellular growth and translational accuracy.

The amino acid sequences of mutationally altered ribosomal protein L7/L12 from four different rplL mutants of Escherichia coli were determined and correlated with some features of the mutant ribosomes. Two of the rplL mutations are deletions around position 40, which give rise to a shortened hinge region between the two domains of L7/L12. The other two mutants harbor point mutations at position 74 (Gly----Asp) or at position 82 (Glu----Lys), which are in or close to an evolutionarily conserved sequence in the C-terminal domain. The two latter mutations are associated with decreased rates of growth and translational elongation. All four mutants show increased nonsense codon read-through in vivo. Ribosomes from one of the deletion mutants show clearly increased missense error rates in vitro.

Amino Acids↗
PubMed1986

Functional interactions in vivo between suppressor tRNA and mutationally altered ribosomal protein S4.

Ribosomal mutants (rpsD) which are associated with a generally increased translational ambiguity were investigated for their effects in vivo on individual tRNA species using suppressor tRNAs as models. It was found that nonsense suppression is either increased, unaffected or decreased depending on the codon context and the rpsD allele involved as well as the nature of the suppressor tRNA. Missense suppression of AGA and AGG by glyT(SuAGA/G) tRNA as well as UGG by glyT(SuUGG-8) tRNA is unaffected whereas suppression of UGG by glyT(SuUGA/G) or glyV(SuUGA/G) tRNA is decreased in the presence of an rpsD mutation. The effects on suppressor tRNA are thus not correlated with the ribosomal ambiguity (Ram) phenotype of the rpsD mutants used in this study. It is suggested that the mutationally altered ribosomes are changed in functional interactions with the suppressor tRNA itself rather than with the competing translational release factor(s) or cognate aminoacyl tRNA. The structure of suppressor tRNA, particularly the anticodon loop, and the suppressed codon as well as the codon context determine the allele specific functional interactions with these ribosomal mutations.

Alleles↗
PubMed1986

Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy.

Effects on translation in vivo by modification deficiencies for 2-methylthio-N6-isopentenyladenosine (ms2i6A) (Escherichia coli) or 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine (ms2io6A) (Salmonella typhimurium) in tRNA were studied in mutant strains. These hypermodified nucleosides are present on the 3' side of the anticodon (position 37) in tRNA reading codons starting with uridine. In E. coli, translational error caused by tRNA was strongly reduced in the case of third-position misreading of a tryptophan codon (UGG) in a particular codon context but was not affected in the case of first-position misreading of an arginine codon (CGU) in another codon context. Misreading of UGA nonsense codons at two different positions was codon context dependent. The efficiencies of some tRNA nonsense suppressors were decreased in a tRNA-dependent manner. Suppressor tRNA which lacks ms2i6A-ms2io6A becomes more sensitive to codon context. Our results therefore indicate that, besides improving translational efficiency, ms2i6A37 and ms2io6A37 modifications in tRNA are also involved in decreasing the intrinsic codon reading context sensitivity of tRNA. Possible consequences for regulation of gene expression are discussed.

Anticodon↗
PubMed1985

Involvement of ribosomal protein L7/L12 in control of translational accuracy.

The effects of two mutations, which map at the rplL locus and both give a changed 50S ribosomal protein L7/L12, were studied. Both mutations are associated with an increased misreading of all three nonsense codons in vivo and ribosomes from the mutants give an increased misreading of the phenylalanine codon UUU by tRNALeu in vitro. The rplL-associated misreading in vitro is not limited to a particular type of mRNA or tRNA. Results from a translational proofreading assay, using mutant ribosomes, suggest that protein L7/L12 is involved in the control of translational accuracy by contributing to the efficiency of a translational proofreading step(s).

Bacterial Proteins↗
PubMed1984

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

A spontaneous mutant was isolated that harbors a weak suppressing activity towards a UAG mutation, together with an inability to grow at 43 degrees 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 without 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.

Chromosomes, Bacterial↗
PubMed1982

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

Three 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.

Alleles↗
PubMed1980

Effects of growth conditions and mutations in RNA polymerase on translational activity in vitro in Escherichia coli.

The translational capacity in vitro in Escherichia coli, using RNA from phage R17 or Q beta as messenger, is several times higher if the extracts are prepared from cells harvested in early exponential phase or grown under conditions of good aeration compared to if extracts are prepared from cells harvested in a later growth phase or grown under semi-aerobic conditions. In low activity extracts the production of phage replicase protein is preferentially affected. Growth of a wild type strain under semi-aerobic conditions has a less pronounced effect on translational capacity in vitro using crude mRNA from normal or T4 infected cells or with poly(U). Mutants were fortuitously found which did not show a lowered translational activity in vitro as a result of entering late phase of growth. Two of these were changed in RNA polymerase. Two different translational inhibitors can be demonstrated in the ribosomal wash fraction obtained from semi-aerobically grown wild type cells, whereas only one was found in the case of aerobically grown cells. The low translational activity of semi-aerobically grown cells in vitro is implied to be dependent on the induction or activation of a translational inhibitor. It behaves like a protein but is not likely to be a protease or RNAse.

Bacteriophages↗
PubMed1980

Analysis of rpsD mutations in Escherichia coli. IV. Accumulation of minor forms of protein S7(K) in ribosomes of rpsD mutant strains due to translational read-through.

A 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.

Amino Acids↗
PubMed1979

Analysis of rpsD mutations in Escherichia coli. I. Comparison of mutants with various alterations in ribosomal protein S4.

Streptomycin-independent revertants were selected from streptomycin-dependent mutants. Twenty-five out of 150 such revertants were temperature sensitive. Ribosomal proteins from 18 temperature-sensitive and 10 temperature-insensitive revertants were analysed by SDS-polyacrylamide gel electrophoresis. Seventeen of the former but none of the latter category showed an alteration of protein S4. The mutated rpsD allele of 6 temperature-sensitive revertants was transduced into a rpsL+ strain. In all cases an increased suppressibility of T4 amber phages was observed. Such suppressibility was not observed in the original rpsD, rpsL strains. All 18 temperature-sensitive mutants were disturbed in the processing of 17s to 16s RNA at non-permissive temperature and the accumulated 17s RNA was degraded. Temperature-insensitive rpsD revertants could be isolated, which had gained a second alteration in S4. Such revertants, which had lost the temperature-sensitive property, were also unable to suppress growth of T4 amber phages. It is concluded that temperature-sensitive growth, inability to process 17s RNA and to assemble 30S ribosomes at non-permissive temperature as well as increased translational ambiguity are highly correlated properties in rpsD mutants.

Bacterial Proteins↗