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. 1974 Feb;71(2):460–463. doi: 10.1073/pnas.71.2.460

An Escherichia coli Mutant with an Altered Elongation Factor Tu

J H Lupker 1, G J Verschoor 1, F W M De Rooij 1, A Rörsch 1, L Bosch 1
PMCID: PMC388026  PMID: 4592692

Abstract

A thermosensitive mutant of Escherichia coli has been isolated that is unable to replicate the bacteriophage MS2 at 42° but permits phage production at 37°. Thermal inactivation studies of the supernatant enzymes show that this mutant contains a factor essential for the polymerization of phenylalanine from phenylalanyl-tRNA that at 50° is more rapidly inactivated than the corresponding wild-type factor. The elongation factor Tu (EF-Tu) was isolated and purified to apparent homogeneity as the EF-Tu·GDP complex, both from mutant and wild-type cells.

Addition of purified wild-type EF-Tu·GDP to reaction mixtures fully restored the activity of thermally inactivated mutant supernatants. These experiments excluded EF-Ts as the thermolabile factor involved. Similar inactivation studies, dealing with the purified factors and performed in reaction mixtures that were not supplemented with GDP, revealed that the half-life of mutant EF-Tu·GDP at 50° was 1.5 min, that of the wild-type factor 6 min. Addition of GDP (10μM) to the medium reduced the inactivation rate of both wild-type and mutant factor and also the difference in inactivation kinetics. Besides the altered elongation factor Tu, the mutant skill contains a second mutation affecting the glutaminyl-tRNA synthetase.

Keywords: protein synthesis, heat inactivation kinetics, GDP

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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