Mutants of EF‐Tu defective in binding aminoacyl‐tRNA

Abstract

Five single amino acid substitution variants of EF-Tu from Salmonella typhimurlum were tested for their ability to promote poly(U)-translation in vitro. The substitutions are Leut2°Gln, Gln124Arg and Tyr 16° (Asp or Asn or Cys). They were selected by their kirromycin resistant phenotypes and all substitutions are in domain I at the interface between domains I and III of the EF-Tu. GTP configuration. The different EF-Tu variants exhibit a spectrum of phenotypes. First, kcat/KM for the interaction between ternary complex and the programmed ribosome is apparently reduced by the substitutions Leu12°Gln, Gln124Arg and Tyrl6°Cys. Second, this reduction is caused by a defect in the interaction between these EF-Tu variants and aminoacyl-tRNA during translation. Third, in four cases out of five the affinity of the complex between EF-Tu" GTP and aminoacyi-tRNA is significantly decreased. The most drastic reduction is observed for the Gln124Arg change, where the association constant is 30-fold lower than in the wild-type case. Fourth, missense errors are increased as well as decreased by the different amino acid substitutions. Finally, the dissociation rate constant (ka) for the release of GDP from EF-Tu is increased 6-fold by the Tyrl6°Cys substitution, but remains unchanged in the four other cases. These results show that the formation of ternary complex is sensitive to many different alterations in the domain I-III interface of EF-Tu.

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

“…One possibility is that the weak affinity of the mutant EF-Tu for aminoacyl-tRNAs [9] exposes aminoacylated-tRNA to an increased rate of deacylation before it can enter into the ternary complex [38]. However, another possibility is suggested by our observation that the level of several different tRNA synthetase transcripts, including proS, is lower in the mutant strain (Figure 4).…”

“…This mutant EF-Tu has a reduced affinity for aa-tRNA but is otherwise proficient in translation in vitro [9].…”

Mutants of the RNA‐processing enzyme RNase E reverse the extreme slow‐growth phenotype caused by a mutant translation factor EF‐Tu

“…The antibiotic kirromycin binds to EF‐Tu and inhibits protein synthesis by preventing EF‐Tu·GDP from leaving the ribosome (Wolf et al ., 1974). Several mutants have been isolated with single‐amino‐acid substitutions in EF‐Tu that make them resistant to kirromycin (Abdulkarim et al ., 1994; 1996). Kirromycin‐resistance is recessive and is expressed in strains carrying resistance mutations in both tuf genes or when the second tuf gene is inactivated (Hughes, 1990; Abdulkarim et al ., 1991).…”

“…Kirromycin‐resistance is recessive and is expressed in strains carrying resistance mutations in both tuf genes or when the second tuf gene is inactivated (Hughes, 1990; Abdulkarim et al ., 1991). Some kirromycin‐resistant mutant variants of EF‐Tu have a reduced affinity for binding aminoacyl‐tRNA (Abdulkarim et al ., 1996). Among these, EF‐Tu with the substitution Gln125Arg in the interface between structural domains I and III (Abdulkarim et al ., 1994) has the most extreme phenotype in vitro , with a 30‐fold reduction in the association constant for aminoacyl‐tRNA (Abdulkarim et al ., 1996).…”

Concerted evolution of duplicate fla genes in Campylobacter

“…This indicates that there are mechanisms to homogenize within a species the sequence of genes that are undergoing concerted evolution. Examples of concerted evolution in bacteria include complete operons such as the rRNA operons (Mattatall et al, 1996), entire genes such as tufA and tufB in Salmonella typhimurium (Abdulkarim & Hughes, 1996) or catIJF and pcaIJF in Acinetobacter calcoaceticus (Kowalchuk et al, 1995), and tandem repeats within genes, such as those seen in alpha C protein of group B streptococci (Madoff et al, 1996).…”