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Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis.

Villet R, Fonvielle M, Busca P, Chemama M, Maillard AP, Hugonnet JE, Dubost L, Marie A, Josseaume N, Mesnage S, Mayer C, Valéry JM, Ethève-Quelquejeu M, Arthur M - Nucleic Acids Res. (2007)

Bottom Line: Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme.In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv).The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U872, LRMA, Centre de Recherche des Cordeliers, Pôle 4, Equipe 12, Paris, F-75006, France.

ABSTRACT
The FemX(Wv) aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNA(Ala) to UDP-MurNAc-pentadepsipeptide. FemX(Wv) is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemX(Wv) is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemX(Wv) depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv). The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

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Semi-synthesis of Ala-tRNAAla analogues. (A) The protected dinucleotide pdCpA-Ala-pentenoyl was ligated to a 74-nt in vitro transcript by the T4 RNA ligase. The product of the reaction was deprotected leading to an acylated-RNA molecule identical to Ala-tRNAAla except for the presence of a 3′deoxycytidine instead of a cytidine at position 75. Nucleotide substitutions were introduced in the 74-nt in vitro transcript using the PCR approach described above except that one of the two mutagenic primers lacked the two 5′ bases specifying bases 75 and 76 of the transcript. (B) Schematic representation of the main steps for synthesis of the dinucleotide pdCpA-Ala-pentenoyl. (C) Analysis of the T4 ligation products by denaturing polyacrylamide electrophoresis. Lane 1, 76-nt transcript with the wild-type tRNAAla sequence; lane 2, 74-nt in vitro transcript; lane 3, ligation of the 74-nt transcript with pdCpA-Ala-pentenoyl.
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Figure 7: Semi-synthesis of Ala-tRNAAla analogues. (A) The protected dinucleotide pdCpA-Ala-pentenoyl was ligated to a 74-nt in vitro transcript by the T4 RNA ligase. The product of the reaction was deprotected leading to an acylated-RNA molecule identical to Ala-tRNAAla except for the presence of a 3′deoxycytidine instead of a cytidine at position 75. Nucleotide substitutions were introduced in the 74-nt in vitro transcript using the PCR approach described above except that one of the two mutagenic primers lacked the two 5′ bases specifying bases 75 and 76 of the transcript. (B) Schematic representation of the main steps for synthesis of the dinucleotide pdCpA-Ala-pentenoyl. (C) Analysis of the T4 ligation products by denaturing polyacrylamide electrophoresis. Lane 1, 76-nt transcript with the wild-type tRNAAla sequence; lane 2, 74-nt in vitro transcript; lane 3, ligation of the 74-nt transcript with pdCpA-Ala-pentenoyl.

Mentions: Detection of UDP-MurNAc-hexapeptide synthesis in the coupled assay used above implies that nucleotide substitutions are tolerated both by the AlaRS and by FemXWv. Thus, the assay provides a rapid method to determine whether nucleotide substitutions are tolerated by FemXWv provided that the modified tRNAAla molecules are substrate of the AlaRS. The assay is highly sensitive to qualitatively detect residual activity since each tRNA molecule can participate to several catalytic cycles (Figure 2). However, the absence of UDP-MurNAc-hexapeptide synthesis in the coupled assay, as observed for the substitutions at positions C72, C71 and G3–U70, may indicate that the modified tRNAs are not substrate of AlaRS although they can be used by FemXWv. Semi-synthesis of Ala-tRNAAla analogues harboring these substitutions was therefore developed to obtain esterified tRNAAla independently from the AlaRS (Figure 7A). This approach was originally developed to introduce non-natural amino acids into proteins by using in vitro coupled transcription-translation systems (30). Briefly, a dinucleotide esterified by an amino acid analogue is obtained by organic synthesis (Figure 7B) and added to an incomplete tRNA lacking the 3′-end CA dinucleotide using the T4 RNA ligase (30). The ligation restores the complete tRNA sequence with a 3′ esterified residue. A 2′ deoxycytidine is incorporated into the dinucleotide to facilitate its synthesis. In preliminary experiments, we generated a FemXWv substrate by this approach which contained the wild-type tRNAAla sequence and l-Ala as the 3′ amino acyl group (Figure 7C). Transfer of l-Ala from this Ala-tRNAAla analogue to UDP-MurNAc-pentapeptide was detected by mass spectrometry indicating that FemXWv tolerates substitution of a ribose by a 2′deoxyribose at position 75.Figure 7.


Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis.

Villet R, Fonvielle M, Busca P, Chemama M, Maillard AP, Hugonnet JE, Dubost L, Marie A, Josseaume N, Mesnage S, Mayer C, Valéry JM, Ethève-Quelquejeu M, Arthur M - Nucleic Acids Res. (2007)

Semi-synthesis of Ala-tRNAAla analogues. (A) The protected dinucleotide pdCpA-Ala-pentenoyl was ligated to a 74-nt in vitro transcript by the T4 RNA ligase. The product of the reaction was deprotected leading to an acylated-RNA molecule identical to Ala-tRNAAla except for the presence of a 3′deoxycytidine instead of a cytidine at position 75. Nucleotide substitutions were introduced in the 74-nt in vitro transcript using the PCR approach described above except that one of the two mutagenic primers lacked the two 5′ bases specifying bases 75 and 76 of the transcript. (B) Schematic representation of the main steps for synthesis of the dinucleotide pdCpA-Ala-pentenoyl. (C) Analysis of the T4 ligation products by denaturing polyacrylamide electrophoresis. Lane 1, 76-nt transcript with the wild-type tRNAAla sequence; lane 2, 74-nt in vitro transcript; lane 3, ligation of the 74-nt transcript with pdCpA-Ala-pentenoyl.
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Figure 7: Semi-synthesis of Ala-tRNAAla analogues. (A) The protected dinucleotide pdCpA-Ala-pentenoyl was ligated to a 74-nt in vitro transcript by the T4 RNA ligase. The product of the reaction was deprotected leading to an acylated-RNA molecule identical to Ala-tRNAAla except for the presence of a 3′deoxycytidine instead of a cytidine at position 75. Nucleotide substitutions were introduced in the 74-nt in vitro transcript using the PCR approach described above except that one of the two mutagenic primers lacked the two 5′ bases specifying bases 75 and 76 of the transcript. (B) Schematic representation of the main steps for synthesis of the dinucleotide pdCpA-Ala-pentenoyl. (C) Analysis of the T4 ligation products by denaturing polyacrylamide electrophoresis. Lane 1, 76-nt transcript with the wild-type tRNAAla sequence; lane 2, 74-nt in vitro transcript; lane 3, ligation of the 74-nt transcript with pdCpA-Ala-pentenoyl.
Mentions: Detection of UDP-MurNAc-hexapeptide synthesis in the coupled assay used above implies that nucleotide substitutions are tolerated both by the AlaRS and by FemXWv. Thus, the assay provides a rapid method to determine whether nucleotide substitutions are tolerated by FemXWv provided that the modified tRNAAla molecules are substrate of the AlaRS. The assay is highly sensitive to qualitatively detect residual activity since each tRNA molecule can participate to several catalytic cycles (Figure 2). However, the absence of UDP-MurNAc-hexapeptide synthesis in the coupled assay, as observed for the substitutions at positions C72, C71 and G3–U70, may indicate that the modified tRNAs are not substrate of AlaRS although they can be used by FemXWv. Semi-synthesis of Ala-tRNAAla analogues harboring these substitutions was therefore developed to obtain esterified tRNAAla independently from the AlaRS (Figure 7A). This approach was originally developed to introduce non-natural amino acids into proteins by using in vitro coupled transcription-translation systems (30). Briefly, a dinucleotide esterified by an amino acid analogue is obtained by organic synthesis (Figure 7B) and added to an incomplete tRNA lacking the 3′-end CA dinucleotide using the T4 RNA ligase (30). The ligation restores the complete tRNA sequence with a 3′ esterified residue. A 2′ deoxycytidine is incorporated into the dinucleotide to facilitate its synthesis. In preliminary experiments, we generated a FemXWv substrate by this approach which contained the wild-type tRNAAla sequence and l-Ala as the 3′ amino acyl group (Figure 7C). Transfer of l-Ala from this Ala-tRNAAla analogue to UDP-MurNAc-pentapeptide was detected by mass spectrometry indicating that FemXWv tolerates substitution of a ribose by a 2′deoxyribose at position 75.Figure 7.

Bottom Line: Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme.In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv).The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U872, LRMA, Centre de Recherche des Cordeliers, Pôle 4, Equipe 12, Paris, F-75006, France.

ABSTRACT
The FemX(Wv) aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNA(Ala) to UDP-MurNAc-pentadepsipeptide. FemX(Wv) is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemX(Wv) is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemX(Wv) depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv). The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

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