Missense substitutions lethal to essential functions of EF-Tu

Abdulkarim, Farhad; Tuohy, Tmf; Buckingham, Richard H.; Hughes, Diarmaid

doi:10.1016/0300-9084(91)90178-4

Cited by 19 publications

(9 citation statements)

References 52 publications

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“…The selection of the mutation studied here, tufB414, has been described previously (1). Briefly (23) by cotransduction with the linked marker zhb-736::TnlO.…”

Section: Methodsmentioning

confidence: 99%

A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome

Tubulekas

Hughes

1993

J Bacteriol

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Elongation factor Tu (EF-Tu) GTP has the primary function of promoting the efficient and correct interaction of aminoacyl-tRNA with the ribosome. Very little is known about the elements in EF-Tu involved in this interaction. We describe a mutant form of EF-Tu, isolated in Salmonella typhimurium, that causes a severe defect in the interaction of the ternary complex with the ribosome. The mutation causes the substitution of Val for Gly-280 in domain II of EF-Tu. The in vivo growth and translation phenotypes of strains harboring this mutation are indistinguishable from those of strains in which the same tufgene is insertionally inactivated. Viable cells are not obtained when the other tuf gene is inactivated, showing that the mutant EF-Tu alone cannot support cell growth. We have confirmed, by partial protein sequencing, that the mutant EF-Tu is present in the cells. In vitro analysis of the natural mixture of wild-type and mutant EF-Tu allows us to identify the major defect of this mutant. Our data shows that the EF-Tu is homogeneous and competent with respect to guanine nucleotide binding and exchange, stimulation of nucleotide exchange by EF-Ts, and ternary complex formation with aminoacyl-tRNA. However various measures of translational efficiency show a significant reduction, which is associated with a defective interaction between the ribosome and the mutant EFTu. GTP aminoacyl-tRNA complex. In addition, the antibiotic kirromycin, which blocks translation by binding EF-Tu on the ribosome, fails to do so with this mutant EF-Tu, although it does form a complex with EF-Tu. Our results suggest that this region of domain II in EF-Tu has an important function and influences the binding of the ternary complex to the codon-programmed ribosome during protein synthesis. Models involving either a direct or an indirect effect of the mutation are discussed.The prokaryotic translation factor elongation factor Tu (EF-Tu) mediates the productive interaction of aminoacyltRNA (aa-tRNA) with the ribosome. The ternary complex EF-Tu. GTP. aa-tRNA interacts with the ribosome such that the anticodon region of aa-tRNA is properly positioned in the ribosomal A site on the 30S subunit, while the aa region remains bound to EF-Tu outside the A site (43). EF-Tu on the ribosome protects bases in the universally conserved a-sarcin loop of 23S rRNA on the 50S ribosomal subunit, suggesting that this may form at least part of its ribosomal binding site (20,44). This conclusion is supported by data showing that a base substitution mutation in the ot-sarcin loop can affect the binding of ternary complex to the ribosome (62). If the tRNA codon and the mRNA anticodon in the 30S A site match, there is a high probability that the GTP on EF-Tu will be hydrolyzed. Whether the probability of GTP hydrolysis depends mainly on the length of time that the ternary complex spends on the ribosome or on signals transmitted to EF-Tu after correct codon-anticodon interaction is an open question. However, after GTP hydrolysis, EF-Tu-GDP leaves the ribosome and is...

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“…The selection of the mutation studied here, tufB414, has been described previously (1). Briefly (23) by cotransduction with the linked marker zhb-736::TnlO.…”

Section: Methodsmentioning

confidence: 99%

A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome

Tubulekas

Hughes

1993

J Bacteriol

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“…In the first experiment we took advantage of a tufB mutation Val226Phe, nucleotide G679T (Abdulkarim et al, 1991), which has a dominant negative effect on growth-rate. This tufB allele does not support cell viability in the absence of another active tuf gene.…”

Section: Sequence Transfer Selected By Growth Ratementioning

confidence: 99%

“…In addition the strains carry as appropriate tufA8 (Ala375Thr), tufB103 (Ala375Thr) or other tuf mutations as mentioned in the text. The isolation of the various tuf mutations used is described elsewhere (Abdulkarim et al, 1991(Abdulkarim et al, , 1994Hughes et al, 1987;Hughes 1987Hughes , 1990. Additional mutations affecting recombination or repair were transduced into these basic strains by selecting for linked (recA1), or inserted (recB, dam, mutS, mutL, mutH, uvrD and mutB), antibiotic resistant markers kindly supplied by Dr Andrew Hessel and Dr Kenneth Sanderson of the Salmonella Genetic Stock Center, Calgary, Canada.…”

Section: Bacterial Strainsmentioning

confidence: 99%

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Homologous Recombination between thetufGenes ofSalmonella typhimurium

Abdulkarim

Hughes

1996

Journal of Molecular Biology

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“…Each gene can be individually inactivated without lethal effect [5]. Strains in which one tuf gene is inactivated produce approximately 66% of the wild-type amount of EF-Tu and have a maximum growth rate in rich medium (Luria broth, LB) that is reduced to a similar degree [2], [3], [6]. Strains in which only one tuf gene is present (or active) facilitate the study of the phenotypes associated with mutant variants of EF-Tu.…”

Section: Introductionmentioning

confidence: 99%

Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu

2014

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Transcription and translation of mRNA's are coordinated processes in bacteria. We have previously shown that a mutant form of EF-Tu (Gln125Arg) in Salmonella Typhimurium with a reduced affinity for aa-tRNA, causes ribosome pausing, resulting in an increased rate of RNase E-mediated mRNA cleavage, causing extremely slow growth, even on rich medium. The slow growth phenotype is reversed by mutations that reduce RNase E activity. Here we asked whether the slow growth phenotype could be reversed by overexpression of a wild-type gene. We identified spoT (encoding ppGpp synthetase/hydrolase) as a gene that partially reversed the slow growth rate when overexpressed. We found that the slow-growing mutant had an abnormally high basal level of ppGpp that was reduced when spoT was overexpressed. Inactivating relA (encoding the ribosome-associated ppGpp synthetase) also reduced ppGpp levels and significantly increased growth rate. Because RelA responds specifically to deacylated tRNA in the ribosomal A-site this suggested that the tuf mutant had an increased level of deacylated tRNA relative to the wild-type. To test this hypothesis we measured the relative acylation levels of 4 families of tRNAs and found that proline isoacceptors were acylated at a lower level in the mutant strain relative to the wild-type. In addition, the level of the proS tRNA synthetase mRNA was significantly lower in the mutant strain. We suggest that an increased level of deacylated tRNA in the mutant strain stimulates RelA-mediated ppGpp production, causing changes in transcription pattern that are inappropriate for rich media conditions, and contributing to slow growth rate. Reducing ppGpp levels, by altering the activity of either SpoT or RelA, removes one cause of the slow growth and reveals the interconnectedness of intracellular regulatory mechanisms.

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Missense substitutions lethal to essential functions of EF-Tu

Cited by 19 publications

References 52 publications

A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome

A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome

Homologous Recombination between thetufGenes ofSalmonella typhimurium

Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu

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