Abstract
Cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) is required for translation and is typically synthesized by cysteinyl-tRNA synthetase (CysRS). However, Methanocaldococcus jannaschii synthesizes Cys-tRNA(Cys) by an indirect pathway, whereby O-phosphoseryl-tRNA synthetase (SepRS) acylates tRNA(Cys) with phosphoserine (Sep), and Sep-tRNA-Cys-tRNA synthase (SepCysS) converts the tRNA-bound phosphoserine to cysteine. We show here that M. jannaschii SepRS differs from CysRS by recruiting the m1G37 modification as a determinant for aminoacylation, and in showing limited discrimination against mutations of conserved nucleotides. Kinetic and binding measurements show that both SepRS and SepCysS bind the reaction intermediate Sep-tRNA(Cys) tightly, and these two enzymes form a stable binary complex that promotes conversion of the intermediate to the product and sequesters the intermediate from binding to elongation factor EF-1alpha or infiltrating into the ribosome. These results highlight the importance of the protein binary complex for efficient synthesis of Cys-tRNA(Cys).