Protein Synthesis in Escherichia coli with Mischarged tRNA

Abstract

ABSTRACT Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA Asp , while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA Asn , a required intermediate in protein synthesis in many organisms (but not in Escherichia coli ). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND- aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA Asn and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA Asn are detrimental to E. coli , smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.