The Conserved Cys-X 1 -X 2 -Cys Motif Present in the TtcA Protein Is Required for the Thiolation of Cytidine in Position 32 of tRNA from Salmonella enterica serovar Typhimurium

Abstract

ABSTRACT The modified nucleoside 2-thiocytidine (s 2 C) has so far been found in tRNA from organisms belonging to the phylogenetic domains Archaea and Bacteria . In the bacteria Escherichia coli and Salmonella enterica serovar Typhimurium, s 2 C is present in position 32 of only four tRNA species— \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{ICG}^{Arg}\) \end{document} , \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{CCG}^{Arg}\) \end{document} , \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{mnm^{5}UCU}^{Arg}\) \end{document} , and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{GCU}^{Ser}\) \end{document} . An in-frame deletion of an S. enterica gene (designated ttcA , for “two-thio-cytidine”) was constructed, and such a mutant has no detectable s 2 C in its tRNA. The TtcA protein family is characterized by the existence of both a PP-loop and a Cys-X 1 -X 2 -Cys motif in the central region of the protein but can be divided into two distinct groups based on the presence and location of additional Cys-X 1 -X 2 -Cys motifs in terminal regions of the sequence. Mutant analysis showed that both cysteines in this central conserved Cys-X 1 -X 2 -Cys motif are required for the formation of s 2 C. The Δ ttcA1 mutant grows at the same rate as the congenic wild-type strain, and no growth disadvantage caused by the lack of s 2 C was observed in a mixed-population experiment. Lack of s 2 C32 did not reduce the selection rate at the ribosomal aminoacyl-tRNA site (A-site) for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(Arg-tRNA_{ICG}^{Arg}\) \end{document} at any of its cognate CGN codons, whereas A-site selection at AGG by \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(Arg-tRNA_{mnm^{5}UCU}^{Arg}\) \end{document} was dependent on the presence of s 2 C32. The presence of s 2 C32 in peptidyl- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{CCU}^{Arg}\) \end{document} or in peptidyl- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{mnm^{5}UCU}^{Arg}\) \end{document} interfered with decoding in the A-site. The presence of s 2 C32 in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{ICG}^{Arg}\) \end{document} decreased the rate of translation of the CGA codon but not that of the CGU codon.