Substrate specificities of Escherichia coli ItaT that acetylates aminoacyl-tRNAs

Abstract

Escherichia coli ItaT toxin reportedly acetylates the α-amino group of the aminoacyl-moiety of Ile-tRNAIle specifically, using acetyl-CoA as an acetyl donor, thereby inhibiting protein synthesis. The mechanism of the substrate specificity of ItaT had remained elusive. Here, we present functional and structural analyses of E. coli ItaT, which revealed the mechanism of ItaT recognition of specific aminoacyl-tRNAs for acetylation. In addition to Ile-tRNAIle, aminoacyl-tRNAs charged with hydrophobic residues, such as Val-tRNAVal and Met-tRNAMet, were acetylated by ItaT in vivo. Ile-tRNAIle, Val-tRNAVal and Met-tRNAMet were acetylated by ItaT in vitro, while aminoacyl-tRNAs charged with other hydrophobic residues, such as Ala-tRNAAla, Leu-tRNALeu and Phe-tRNAPhe, were less efficiently acetylated. A comparison of the structures of E. coli ItaT and the protein N-terminal acetyltransferase identified the hydrophobic residues in ItaT that possibly interact with the aminoacyl moiety of aminoacyl-tRNAs. Mutations of the hydrophobic residues of ItaT reduced the acetylation activity of ItaT toward Ile-tRNAIlein vitro, as well as the ItaT toxicity in vivo. Altogether, the size and shape of the hydrophobic pocket of ItaT are suitable for the accommodation of the specific aminoacyl-moieties of aminoacyl-tRNAs, and ItaT has broader specificity toward aminoacyl-tRNAs charged with certain hydrophobic amino acids.

Mechanism of aminoacyl-tRNA acetylation by an aminoacyl-tRNA acetyltransferase AtaT from enterohemorrhagic E. coli

“…1b) 13 , there is no direct evidence for Met-tRNAf Met acetylation by AtaT in vivo. To evaluate the in vivo target of aminoacyl-tRNAs for acetylation by AtaT, AtaT was expressed in E. coli and the intracellular aminoacyl-tRNAs were analyzed by LC/MS 26 (Fig. 4a).…”

“…GNAT family toxins were reported to have different specificities for aminoacyl-tRNAs [12][13][14][15][16] . Recently, ItaT from E. coli HS was shown to acetylate Ile-tRNA Ile , Val-tRNA Val , and Met-tRNA Met in vivo and in vitro 26 . Since there is no consensus sequence in the acceptor stem of the substrate isoaccepting tRNAs, ItaT presumably selects its substrate aminoacyl-tRNA by mainly recognizing the aminoacyl moieties of aminoacyl-tRNAs, and the size and shape of the hydrophobic pocket of ItaT are suitable for the accommodation of specific aminoacyl moieties 26 .…”

“…Recently, ItaT from E. coli HS was shown to acetylate Ile-tRNA Ile , Val-tRNA Val , and Met-tRNA Met in vivo and in vitro 26 . Since there is no consensus sequence in the acceptor stem of the substrate isoaccepting tRNAs, ItaT presumably selects its substrate aminoacyl-tRNA by mainly recognizing the aminoacyl moieties of aminoacyl-tRNAs, and the size and shape of the hydrophobic pocket of ItaT are suitable for the accommodation of specific aminoacyl moieties 26 . The hydrophobic pocket of AtaT for the aminoacyl-moieties of aminoacyl-tRNAs would be larger than that of ItaT and suitable for the accommodation of bulkier hydrophobic aromatic residues.…”

“…The DNA fragment encoding glutaminyl-tRNA synthetase, glnS, was PCR amplified from the E. coli genome and cloned into the NdeI and XhoI sites of pET-22b. The plasmids for expression of methionyl-tRNA synthetase and isoleucyl-tRNA synthetase were described previously 26 . The plasmids for the overexpression of other aminoacyl-tRNA synthetases (ARSs: AlaRS, ValRS, LeuRS, GlyRS, TrpRS, TyrRS, and PheRS) were kind gifts from Dr. Shimizu (RIKEN, Japan).…”

“…DNA fragment sequences are listed in Supplementary Table 2. The pBSTNAV3 plasmids encoding tRNAf Met , tRNAm Met , tRNAf Met GCU, tRNAf Met UAC, tRNA Leu , tRNA Ile , tRNA Val , and tRNA Ala were described previously 22,23,26 . The E. coli JM101Tr strain was transformed with the pBSTNAV3 plasmid and cultured in 2 × YT medium containing 50 μg mL −1 ampicillin, at 37°C for 24 h. The total tRNA fraction was prepared as described, with modifications 22,34 .…”