Studies on Polypeptide‐Chain‐Elongation Factors from an Extreme Thermophile, Thermus thermophilus HB8

Abstract

Catalytic properties of the elongation factors from Thermus thermophilus HB8 have been studied and compared with those of the factors from Escherichia coli.

Cross‐reactivity studies on the interaction between elongation factors Tu and Ts from Streptomyces aureofaciens and Escherichia coli in the GDP exchange reaction

“…The effects of EF-Ts on these GDP exchange reactions were studied under equilibrium and non-equilibrium conditions. Similar studies have been performed with elongation factors from Caufobacter crescentus [5] and two thermophilic bacteria [6,7]. In all these studies EF-Ts from E. coli did not catalyze GDP exchange on any of these heterologous EF-Tus.…”

Mutations in the G-domain of Elongation Factor G fromThermus thermophilus Affect Both Its Interaction with GTP and Fusidic Acid

“…Moreover, the addition of the ribosomes to EF-G and a GTP analog decreases the dissociation constant of the analog from the complex, which was found to be 59 nM (32). This K d value is ϳ170 times Ͻ10 M for the GTP interaction with E. coli EF-G without ribosomes (21). Thus, the increased affinity of EF-G for GTP analog caused by the ribosomes is paralleled by the effect of FA-hypersensitive mutations in EF-G described here.…”

“…Poly(U)-directed Translation-EF-G from thermophilic microorganisms can substitute their counterpart from E. coli in heterologous cell-free translation system with E. coli ribosomes (17,18,21). EF-G species with the point mutations T84A, D109K, and E119K are active in poly(U)-directed translation (Fig.…”

“…GMP-PNP binding with mutated proteins (300 pmol) was measured by the displacement of [ 3 H]GDP bound to the factor with increasing concentrations of unlabeled GMP-PNP. The concentration of [ 3 H]GDP in the mixture was 0.5 M and that of GMP-PNP varied from 0.5 to 500 M. The dissociation constant for GMP-PNP with EF-G was calculated from titration curves as a concentration of GMP-PNP necessary to displace half of GDP bound to the factor (21).…”

“…Nucleotide-dependent Interactions-GDP interactions with EF-G were tested according to the procedure used for the factors from T. thermophilus (21). The GDP dissociation constant was determined in the reaction mixture (0.2 ml) in 50 mM Tris-HCl buffer, pH 7.6, 10 mM MgCl 2 , 1 mM dithiothreitol , and 70 mM NH 4 Cl containing 300 pmol of EF-G by titration with [ 3 H]GDP (1.1 Ci/mmol) at 37°C.…”