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
Authors: Jäger Gunilla , Leipuviene Ramune , Pollard Michael G. , Qian Qiang , Björk Glenn R. 
Published: January 2004

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—

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,

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,

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, and

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. 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

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at any of its cognate CGN codons, whereas A-site selection at AGG by

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was dependent on the presence of s
2
C32. The presence of s
2
C32 in peptidyl-

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or in peptidyl-

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interfered with decoding in the A-site. The presence of s
2
C32 in

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decreased the rate of translation of the CGA codon but not that of the CGU codon.