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Leucine-specific domain modulates the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase.

Yan W, Tan M, Eriani G, Wang ED - Nucleic Acids Res. (2013)

Bottom Line: Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding.Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD.The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Biology, Center for RNA Research, Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, PR China.

ABSTRACT
The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNA(Leu) recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

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Effect of LSD mutations on tRNA-independent pre-transfer editing. (A) Total editing activity was measured using the AMP formation assay with 0.2 µM EcLeuRS-GKDG in the absence or presence of 5 µM EctRNALeu and 15 mM Nva. (B) A similar assay was performed with 1 µM MmLeuRS-CP1/LSD in the absence or presence of 5 µM EctRNALeu and 15 mM Nva. (C) Contributions of the different editing pathways for each protein: left, sum of the kobs of different editing pathways; right, relative contributions of each pathway. Percentages were calculated from kobs values of AMP formation reported in Table 1. tRNA-independent pre-transfer editing was measured in the absence of tRNA. tRNA-dependent editing was deduced by subtracting the tRNA-independent pre-transfer editing from total editing.
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gkt185-F2: Effect of LSD mutations on tRNA-independent pre-transfer editing. (A) Total editing activity was measured using the AMP formation assay with 0.2 µM EcLeuRS-GKDG in the absence or presence of 5 µM EctRNALeu and 15 mM Nva. (B) A similar assay was performed with 1 µM MmLeuRS-CP1/LSD in the absence or presence of 5 µM EctRNALeu and 15 mM Nva. (C) Contributions of the different editing pathways for each protein: left, sum of the kobs of different editing pathways; right, relative contributions of each pathway. Percentages were calculated from kobs values of AMP formation reported in Table 1. tRNA-independent pre-transfer editing was measured in the absence of tRNA. tRNA-dependent editing was deduced by subtracting the tRNA-independent pre-transfer editing from total editing.

Mentions: In the next experiments, a series of insertion mutants was constructed to mimic a possible evolutionary process. Chimeric proteins were constructed based on the MmLeuRS scaffold. First, the LSD of EcLeuRS was inserted in place of the tetrapeptide GKDG in the MmLeuRS (MmLeuRS-LSD). The resulting MmLeuRS-LSD mutant did not exhibit any detectable aminoacylation activity (data not shown). MmLeuRS-CP1 was constructed by inserting the CP1 domain of EcLeuRS into MmLeuRS, and this chimeric enzyme had both aminoacylation and editing activities (17). When MmLeuRS-CP1 was used as a scaffold to fuse the LSD of EcLeuRS into its catalytic core, the resulting chimera (MmLeuRS-CP1/LSD) had comparable aminoacylation activity to the native MmLeuRS but demonstrated better catalytic efficiency due to greater affinity with tRNA as indicated by a decrease in Km (Table 1). However, the LSD insertion severely decreased the tRNA-independent pre-transfer editing of MmLeuRS and MmLeuRS-CP1, and the observed rate constant for AMP formation in the presence of Nva (an analogue of Leu) dropped from 0.16 and 0.12 to 0.037 s−1 (Table 2). In the presence of EctRNALeu and Nva, the observed rate constant of MmLeuRS-CP1/LSD for AMP formation was comparable with that of MmLeuRS-CP1, and the rate was 3.6-fold (0.61 s−1) greater than that of MmLeuRS (0.17 s−1). This shows that the tRNA-dependent editing pathway became the main editing pathway of MmLeuRS-CP1/LSD, contributing to 94% of the total editing activity [(0.61 − 0.037)/0.61], whereas the corresponding value in MmLeuRS-CP1 was just 78% [(0.55 − 0.12)/0.55] (Figure 2C). On the other hand, when the LSD of EcLeuRS was replaced by the GKDG tetrapeptide of MmLeuRS to form EcLeuRS-GKDG, the observed rate constant for AMP formation in the presence of Nva of the mutant was 0.75 s−1 compared with 0.33 s−1 for the native EcLeuRS (Table 2), indicating that the tRNA-independent pre-transfer editing of EcLeuRS-GKDG contributed much more to total editing (28%; 0.75/2.69) than that of EcLeuRS (9.6%; 0.33/3.42).Figure 2.


Leucine-specific domain modulates the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase.

Yan W, Tan M, Eriani G, Wang ED - Nucleic Acids Res. (2013)

Effect of LSD mutations on tRNA-independent pre-transfer editing. (A) Total editing activity was measured using the AMP formation assay with 0.2 µM EcLeuRS-GKDG in the absence or presence of 5 µM EctRNALeu and 15 mM Nva. (B) A similar assay was performed with 1 µM MmLeuRS-CP1/LSD in the absence or presence of 5 µM EctRNALeu and 15 mM Nva. (C) Contributions of the different editing pathways for each protein: left, sum of the kobs of different editing pathways; right, relative contributions of each pathway. Percentages were calculated from kobs values of AMP formation reported in Table 1. tRNA-independent pre-transfer editing was measured in the absence of tRNA. tRNA-dependent editing was deduced by subtracting the tRNA-independent pre-transfer editing from total editing.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3643597&req=5

gkt185-F2: Effect of LSD mutations on tRNA-independent pre-transfer editing. (A) Total editing activity was measured using the AMP formation assay with 0.2 µM EcLeuRS-GKDG in the absence or presence of 5 µM EctRNALeu and 15 mM Nva. (B) A similar assay was performed with 1 µM MmLeuRS-CP1/LSD in the absence or presence of 5 µM EctRNALeu and 15 mM Nva. (C) Contributions of the different editing pathways for each protein: left, sum of the kobs of different editing pathways; right, relative contributions of each pathway. Percentages were calculated from kobs values of AMP formation reported in Table 1. tRNA-independent pre-transfer editing was measured in the absence of tRNA. tRNA-dependent editing was deduced by subtracting the tRNA-independent pre-transfer editing from total editing.
Mentions: In the next experiments, a series of insertion mutants was constructed to mimic a possible evolutionary process. Chimeric proteins were constructed based on the MmLeuRS scaffold. First, the LSD of EcLeuRS was inserted in place of the tetrapeptide GKDG in the MmLeuRS (MmLeuRS-LSD). The resulting MmLeuRS-LSD mutant did not exhibit any detectable aminoacylation activity (data not shown). MmLeuRS-CP1 was constructed by inserting the CP1 domain of EcLeuRS into MmLeuRS, and this chimeric enzyme had both aminoacylation and editing activities (17). When MmLeuRS-CP1 was used as a scaffold to fuse the LSD of EcLeuRS into its catalytic core, the resulting chimera (MmLeuRS-CP1/LSD) had comparable aminoacylation activity to the native MmLeuRS but demonstrated better catalytic efficiency due to greater affinity with tRNA as indicated by a decrease in Km (Table 1). However, the LSD insertion severely decreased the tRNA-independent pre-transfer editing of MmLeuRS and MmLeuRS-CP1, and the observed rate constant for AMP formation in the presence of Nva (an analogue of Leu) dropped from 0.16 and 0.12 to 0.037 s−1 (Table 2). In the presence of EctRNALeu and Nva, the observed rate constant of MmLeuRS-CP1/LSD for AMP formation was comparable with that of MmLeuRS-CP1, and the rate was 3.6-fold (0.61 s−1) greater than that of MmLeuRS (0.17 s−1). This shows that the tRNA-dependent editing pathway became the main editing pathway of MmLeuRS-CP1/LSD, contributing to 94% of the total editing activity [(0.61 − 0.037)/0.61], whereas the corresponding value in MmLeuRS-CP1 was just 78% [(0.55 − 0.12)/0.55] (Figure 2C). On the other hand, when the LSD of EcLeuRS was replaced by the GKDG tetrapeptide of MmLeuRS to form EcLeuRS-GKDG, the observed rate constant for AMP formation in the presence of Nva of the mutant was 0.75 s−1 compared with 0.33 s−1 for the native EcLeuRS (Table 2), indicating that the tRNA-independent pre-transfer editing of EcLeuRS-GKDG contributed much more to total editing (28%; 0.75/2.69) than that of EcLeuRS (9.6%; 0.33/3.42).Figure 2.

Bottom Line: Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding.Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD.The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Biology, Center for RNA Research, Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, PR China.

ABSTRACT
The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNA(Leu) recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

Show MeSH
Related in: MedlinePlus