Impaired in vitro kinetics of EF‐Tu mutant Aa

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Abstract
The kirromycin-resistant EF-Tu mutant Aa, previously shown to be an antisuppressor for nonsense and missense suppressor tRNAs, has been characterised in a poly(U)-primed translation system in vitro. Two major defects were found in the function of the mutant. First, the dissociation constant for Aa binding to Phe-tRNAPhe was increased tenfold compared to wild-type EF-Tu. Second, kcat/Km for the interaction between the EF-Tu .cntdot. GTP .cntdot. aa-tRNA complex and the ribosome was decreased by the mutation to one third of its wild-type value. No differences were observed between mutant and wild-type factor in the regeneration of EF-Tu .cntdot. GDP from EF-Tu .cntdot. GDP via EF-Ts or in the mistranslation frequency by Leu-tRNA4Leu. The relation between the in vitro results and the mutant phenotype in vivo is discussed.