Maturation of precursor transfer
RNA (pre-tRNA) includes excision of the
5' leader and
3' trailer sequences, removal of
introns and addition of the CCA terminus.
Nucleotide modifications are incorporated at different stages of tRNA processing, after the
RNA molecule adopts the proper conformation. In
bacteria, tRNA(Ile2)
lysidine synthetase (TilS) modifies
cytidine into
lysidine (L; 2-lysyl-cytidine) at the first
anticodon of tRNA(Ile2) (refs 4-9). This modification switches tRNA(Ile2) from a methionine-specific to an isoleucine-specific tRNA. However, the
aminoacylation of tRNA(Ile2) by methionyl-tRNA
synthetase (MetRS), before the modification by TilS, might lead to the misincorporation of
methionine in response to
isoleucine codons. The mechanism used by
bacteria to avoid this pitfall is unknown. Here we show that the TilS
enzyme specifically recognizes and modifies tRNA(Ile2) in its
precursor form, thereby avoiding translation errors. We identified the
lysidine modification in pre-tRNA(Ile2) isolated from RNase-E-deficient
Escherichia coli and did not detect mature tRNA(Ile2) lacking this modification. Our kinetic analyses revealed that TilS can modify both types of
RNA molecule with comparable efficiencies.
X-ray crystallography and mutational analyses revealed that TilS specifically recognizes the entire L-shape structure in pre-tRNA(Ile2) through extensive interactions coupled with sequential domain movements. Our results demonstrate how TilS prevents the recognition of tRNA(Ile2) by MetRS and achieves high specificity for its
substrate. These two key points form the basis for maintaining the fidelity of
isoleucine codon translation in
bacteria. Our findings also provide a rationale for the necessity of incorporating specific modifications at the precursor level during tRNA biogenesis.
