Biomedical subjects

L A Isaksson

Publications and source records attributed to L A Isaksson.

At least 37 records · Page 2Linked to original sources

PubMed1996

Structure of the C-terminal end of the nascent peptide influences translation termination.

The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.

Amino Acid Sequence↗

PubMed1996

Influence of the last amino acid in the nascent peptide on EF-Tu during decoding.

The last two amino acids of the nascent peptide at the ribosomal P-site influence the efficiency of termination readthrough at the stop codon UGA (Mottagui-Tabar et al (1994) EMBO J 13, 249-257; Björnsson et al (1996) EMBO J 15, 1696-1704). Here we analyze this effect on readthrough by wild type or a UGA suppressor form (Su9) of tRNA(Trp) by varying the codons at positions-1 and -2 at the 5' side of UGA. Strains with wild-type or mutant (ArBr) forms of elongation factor Tu (EF-Tu) were analyzed (Vijgenboom et al (1985) EMBO J4, 1049-1052). The effect on readthrough by changing these-1 and -2 codons is different on the two forms of tRNA(Trp) and is also dependent on the structure of EF-Tu. Readthrough by the tRNA(Trp)-derived suppressor, but not wild-type tRNA(Trp), is sensitive to the van der Waals volume of the last amino acid in the nascent peptide. Together with mutant EF-Tu, both forms of tRNA(Trp) are sensitive. The data suggest that the C-terminal amino acid in the nascent peptide is in a functional interaction with the EF-Tu ternary complex. This interaction is changed by mutation in tRNA(Trp) at position 24 or in EF-Tu at position 375. No indication of a changed interaction between the mutant EF-Tu and the penultimate amino acid could be found. Mutant forms of RF2 (Mikuni et al (1991) Biochimie 73, 1509-1516) and ribosomal proteins S4 and S12 (Fáxen et al (1988) J Bacteriol 170, 3756-3760) were found not be altered in sensitivity to the last two amino acids in the nascent peptide.

Codon↗

PubMed1996

Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2.

Escherichia coli 397c is temperature sensitive for growth at 43.5 degrees C and unable to plate bacteriophage P2 at 33 degrees C. The mutation conferring these phenotypes was mapped to the rpoC gene. RNA synthesis is temperature sensitive in the mutant strain, and the beta' subunit of RNA polymerase isolated from this strain exhibits increased electrophoretic mobility. DNA sequence analysis revealed that the mutation is a deletion of 16 bp, resulting in a frameshift that leads to truncation of the beta' subunit at the carboxy terminus.

Amino Acid Sequence↗

PubMed1994

Starvation in vivo for aminoacyl-tRNA increases the spatial separation between the two ribosomal subunits.

Structures in situ of individual ribosomes in E. coli have been determined by computer-aided electron microscope tomography using a tilt series of positively stained embedded cellular sections. Amino acid starvation of a bacterial culture, causing a deficiency for aminoacyl-tRNA, induces a spatial separation between the ribosomal subunits compared with ribosomes in exponentially growing cells. Eight ribosomes from each growth condition were aligned to each other, and the two average structures were determined. Comparison of these suggests that the distance between the two subunits increases by approximately 3 nm upon starvation for aminoacyl-tRNA during protein synthesis. Ribosomes in most other states of the translational elongation cycle in exponentially growing cells show a more compact structure than previously realized.

Amino Acids↗

PubMed1994

Functional interactions between translation, transcription and ppGpp in growing Escherichia coli.

Strains with a relA mutation together with three different alleles of spoT were used to study the effects of different levels of ppGpp on production time for beta-galactosidase, transcriptional polarity and readthrough of a stop codon by near-cognate tRNA or a suppressor tRNA. The influences of an rpsL(S12) allele and a miaA mutation, together giving decreased efficiency of translation, as well as an rpoB mutation, coding for an altered RNA polymerase, were also investigated. The spoT alleles which give total deficiency for ppGpp, or a level which is increased several-fold (Sarubbi et al. (1988) Mol. Gen. Genet. 213, 214-222), had at the most a marginal effect on the production time for a beta-galactosidase molecule or translational misreading of a nonsense mutation. The efficiency of an amber tRNA suppressor is not affected by ppGpp in strains with an otherwise wildtype translational machinery. These data suggest that ppGpp does not influence directly the translational process in vivo. Instead, ppGpp is found to interfere with transcriptional readthrough in a manner which is dependent on the rpsL224, miaA, as well as the rpoB mutations. Similarly, bacterial growth is affected by ppGpp in a manner which is dependent on properties of both the transcriptional and translational apparatus together. It is suggested that the primary effect of ppGpp is on transcriptional readthrough, but this effect is modified by translational/transcriptional coupling.

DNA-Directed RNA Polymerases↗

PubMed1994

Genetic implication for an interaction between release factor one and ribosomal protein L7/L12 in vivo.

Strains with mutant alleles of the genes encoding release factor one (RF1), prfA, and ribosomal protein L7/L12, rplL, have been analyzed. The prfA1 allele has previously been shown to be associated with temperature sensitivity for growth at 43 degrees C and increased misreading of the nonsense codons UAG and UAA. Here we show that the prfA1 mutation is a nucleotide substitution that results in an arginine to proline change at amino acid position 137 in RF1. This alters the factor in a domain which may be involved in ribosome-binding. The rplL564 allele codes for a mutant form of protein L7/L12 that has a change in its conserved, translation-factor binding, domain. The rplL564 mutation suppresses the temperature sensitive phenotype and the increased translational misreading associated with the prfA1 allele, while other studied rplL mutant alleles do not. These data are consistent with an interaction between L7/L12 and RF1 in vivo.

Bacterial Proteins↗

PubMed1994

Antisuppression by a mutation in rpsM(S13) giving a shortened ribosomal protein S13.

The phenotype associated with an rpsM(S13) mutation, originally isolated in Escherichia coli in a selection for pseudoreversion of streptomycin dependence, was studied in strains lacking the original mutations for antibiotic dependence. The rpsM mutation gives a decreased translational step time and a reduced growth rate. It functions as a strong antisuppressor to both the serU(Su1) amber suppressor and the trpT(Su9) opal suppressor, whereas the tyrT(Su3) amber suppressor is much less affected. The small ribosomal subunit from the rpsM mutant shows a reduced sedimentation coefficient but is able to form apparently normal 70S ribosomes as judged by ultracentrifugational analysis. Cloning and sequencing show that the rpsM mutation is a CAG to TAG alteration at codon position 100, giving an S13 protein which is shortened by 19 amino acids at its C-terminal end. This implies that the C-terminal domain of the protein that is involved in binding to 16S ribosomal RNA should be affected.

Amino Acid Sequence↗

PubMed1994

The second to last amino acid in the nascent peptide as a codon context determinant.

Forty-two different sense codons, coding for all 20 amino acids, were placed at the ribosomal E site location, two codons upstream of a UGA or UAG codon. The influence of these variable codons on readthrough of the stop codons was measured in Escherichia coli. A 30-fold difference in readthrough of the UGA codon was observed. Readthrough is not related to any property of the upstream codon, its cognate tRNA or the nature of its codon-anticodon interaction. Instead, it is the amino acid corresponding to the second upstream codon, in particular the acidic/basic property of this amino acid, which seems to be a major determinant. This amino acid effect is influenced by the identity of the A site stop codon and the efficiency of its decoding tRNA, which suggests a correlation with ribosomal pausing. The magnitude of the amino acid effect is in some cases different when UGA is decoded by a wildtype form of tRNA(Trp) as compared with a suppressor form of the same tRNA. This indicates that the structure of the A site decoding tRNA is also a determinant for the amino acid effect.

Amino Acids↗

PubMed1993

UGA codon context which spans three codons. Reversal by ms2i6A37 in tRNA, mutation in rpsD(S4) or streptomycin.

Mutant UGA codon contexts which previously have been identified at different positions in the lacI part of a fused lacIlacZ gene were characterized with respect to translational readthrough in another genetic surrounding at a constant location. Although readthrough levels are systematically higher in this new location the "tight/leaky" characteristics of these codon contexts are essentially fully determined by the two codons flanking the nonsense codon itself. Analysis of some UGA hybrid contexts shows that the contribution to the codon context character by the codon either at the 5'-side (CCA or AGC) or at the 3'-side (NGU) is independent of the nature of the codon at the other side of UGA if this codon is decoded by trpT(Su9) suppressor tRNA. In a trpT(Su9), miaA double mutant strain, which lacks the ms2i6A37 modification in this tRNA, suppression is decreased at all UGA contexts investigated. However, in one case the contribution to the codon context character by the determinant flanking at one side is negatively affected by the nature of the codon at the other side of UGA. Thus, the character of a nonsense codon context in this case results from both flanking codons acting in a co-operative manner with the tRNA reading the middle UGA codon. This negative context effect is counteracted by a rpsD12 (ribosomal protein S4) mutation or by a sublethal concentration of streptomycin in the growth medium. It is suggested that the ms2i6A37 base in trpT(Su9) suppressor tRNA increases the efficiency of this tRNA by protecting it from ribosomal proofreading which is induced by codon context.

Bacterial Proteins↗

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↗