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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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Residual (%) UDP-MurNAc-hexapeptide synthesis in the AlaRS-FemXWv coupled assay with derivatives of tRNAAla (76-nt) harboring single base substitutions in the acceptor stem. The standard assay was performed with AlaRS (800 nM), FemXWv (500 nM), l-[14C]Ala (50 µM) and the different tRNAs (ca. 5.0 µM). The reaction was incubated for 2 h at 30°C. Under these conditions, total l-[14C]Ala (0.5 nmol) was incorporated into UDP-MurNAc-hexapeptide when RNA with the wild-type sequence was used (100%). ND, not detected.
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Figure 5: Residual (%) UDP-MurNAc-hexapeptide synthesis in the AlaRS-FemXWv coupled assay with derivatives of tRNAAla (76-nt) harboring single base substitutions in the acceptor stem. The standard assay was performed with AlaRS (800 nM), FemXWv (500 nM), l-[14C]Ala (50 µM) and the different tRNAs (ca. 5.0 µM). The reaction was incubated for 2 h at 30°C. Under these conditions, total l-[14C]Ala (0.5 nmol) was incorporated into UDP-MurNAc-hexapeptide when RNA with the wild-type sequence was used (100%). ND, not detected.

Mentions: To facilitate site-directed mutagenesis of the DNA template for in vitro transcription of tRNAAla by T7 RNA polymerase, we have developed a method based on PCR amplification using mutagenic primers. In the classical method (21), the 5′-end of the DNA template that determines the 3′-end of the run off transcript is generated by digestion of a plasmid by the restriction endonuclease BstNI. In this study, the 5′-end of a primer used for PCR amplification of the DNA template provided an alternate way to define the 3′-end of the tRNA transcript. Twenty-four mutagenic primers were used to introduce all possible single nucleotide substitutions in positions C69 to A76 of the acceptor stem. The 5′-end of the in vitro transcript was defined by the localization of the T7 promoter, which was incorporated at the 5′-end of the second primer used for PCR amplification. The mutagenic primers also contained the sequence corresponding to bases 1 to 22 of tRNAAla, thereby allowing the introduction of all possible single nucleotide substitutions in positions 1 to 4. The plasmid template used for PCR amplification did not contain the T7 promoter sequence. Thus, the wild-type tRNAAla sequence could not originate from transcription of the plasmid template. In preliminary experiments, we compared tRNAAla with the wild-type sequence obtained by in vitro transcription of DNA matrices generated by PCR or by the classical digestion of a plasmid template with BstNI. The two types of tRNAAla preparations were equivalent in conditions where the tRNA substrate and FemXWv were rate limiting (data not shown). We also checked that the fidelity of the DNA polymerase used for the PCR was sufficient to avoid false positives. This was established by showing that several substitutions abolished formation of the UDP-MurNAc-hexapeptide product of FemXWv in the coupled assay (Figure 5, see subsequently).Figure 5.


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)

Residual (%) UDP-MurNAc-hexapeptide synthesis in the AlaRS-FemXWv coupled assay with derivatives of tRNAAla (76-nt) harboring single base substitutions in the acceptor stem. The standard assay was performed with AlaRS (800 nM), FemXWv (500 nM), l-[14C]Ala (50 µM) and the different tRNAs (ca. 5.0 µM). The reaction was incubated for 2 h at 30°C. Under these conditions, total l-[14C]Ala (0.5 nmol) was incorporated into UDP-MurNAc-hexapeptide when RNA with the wild-type sequence was used (100%). ND, not detected.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Residual (%) UDP-MurNAc-hexapeptide synthesis in the AlaRS-FemXWv coupled assay with derivatives of tRNAAla (76-nt) harboring single base substitutions in the acceptor stem. The standard assay was performed with AlaRS (800 nM), FemXWv (500 nM), l-[14C]Ala (50 µM) and the different tRNAs (ca. 5.0 µM). The reaction was incubated for 2 h at 30°C. Under these conditions, total l-[14C]Ala (0.5 nmol) was incorporated into UDP-MurNAc-hexapeptide when RNA with the wild-type sequence was used (100%). ND, not detected.
Mentions: To facilitate site-directed mutagenesis of the DNA template for in vitro transcription of tRNAAla by T7 RNA polymerase, we have developed a method based on PCR amplification using mutagenic primers. In the classical method (21), the 5′-end of the DNA template that determines the 3′-end of the run off transcript is generated by digestion of a plasmid by the restriction endonuclease BstNI. In this study, the 5′-end of a primer used for PCR amplification of the DNA template provided an alternate way to define the 3′-end of the tRNA transcript. Twenty-four mutagenic primers were used to introduce all possible single nucleotide substitutions in positions C69 to A76 of the acceptor stem. The 5′-end of the in vitro transcript was defined by the localization of the T7 promoter, which was incorporated at the 5′-end of the second primer used for PCR amplification. The mutagenic primers also contained the sequence corresponding to bases 1 to 22 of tRNAAla, thereby allowing the introduction of all possible single nucleotide substitutions in positions 1 to 4. The plasmid template used for PCR amplification did not contain the T7 promoter sequence. Thus, the wild-type tRNAAla sequence could not originate from transcription of the plasmid template. In preliminary experiments, we compared tRNAAla with the wild-type sequence obtained by in vitro transcription of DNA matrices generated by PCR or by the classical digestion of a plasmid template with BstNI. The two types of tRNAAla preparations were equivalent in conditions where the tRNA substrate and FemXWv were rate limiting (data not shown). We also checked that the fidelity of the DNA polymerase used for the PCR was sufficient to avoid false positives. This was established by showing that several substitutions abolished formation of the UDP-MurNAc-hexapeptide product of FemXWv in the coupled assay (Figure 5, see subsequently).Figure 5.

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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