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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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Mutations in MmtRNALeuUAG that impact editing activity. (A) Cloverleaf structure of MmtRNALeuUAG showing the mutations tested during the study. (B) AMP formation assay in the presence of 15 mM Nva catalyzed by 1 µM MmLeuRS-CP1/LSD in the presence of 5 µM wild-type MmtRNALeuUAG, C20U and C20U + V-arm-5 nt. (C) Graphical representations of AMP formation as a function of time. kobs values of AMP formation were calculated from the slopes, and these are shown in Table 4.
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gkt185-F3: Mutations in MmtRNALeuUAG that impact editing activity. (A) Cloverleaf structure of MmtRNALeuUAG showing the mutations tested during the study. (B) AMP formation assay in the presence of 15 mM Nva catalyzed by 1 µM MmLeuRS-CP1/LSD in the presence of 5 µM wild-type MmtRNALeuUAG, C20U and C20U + V-arm-5 nt. (C) Graphical representations of AMP formation as a function of time. kobs values of AMP formation were calculated from the slopes, and these are shown in Table 4.

Mentions: To identify the structural determinants of tRNALeu responsible for LeuRS ability to discriminate tRNALeus from various species, we compared the tRNALeu sequences from E. coli and M. mobile and focused our attention on three differences between them: (i) the sixth base-pair in the acceptor stem of MmtRNALeuUAG is a wobble base pair (A6•••C67), whereas it is a Watson Crick base pair G6–C67 in EctRNALeuGAG; (ii) the loop of the V-arm of MmtRNALeuUAG contains three nucleotides; however, EctRNALeuGAG has a 4-nucleotide loop; (iii) nucleotide 20, located in the ‘variable pocket’ (20) of the D-loop, is always a U in EctRNALeus but always a C in MmtRNALeuUAG (Figure 3A). Therefore, a series of mutants of MmtRNALeuUAG was constructed. Firstly, the A6•••C67 pair was mutated to a Watson Crick base pair by introducing A6G or C67U mutations. Secondly, in the ‘variable pocket’, nucleotide C20 was changed to a U. Thirdly, the loop of the V-arm was enlarged from three nucleotides to four (V-arm-4 nt) or five (V-arm-5 nt), which are usual sizes for these loops in tRNALeus. MmLeuRS-CP1/LSD leucylated the C20U and V-arm-5 nt mutants at more than twice the catalytic efficiency of the wild-type MmtRNALeuUAG (from 0.3 to 0.75 and 0.70 s−1 µM−1, respectively). MmLeuRS-CP1/LSD leucylated the double mutant (C20U + V-arm-5 nt), where the C20U mutation and V-arm-5 nt mutation were present, and catalytic efficiency (kcat/Km 1.1 s−1 µM−1) (Table 3) almost reached the level of EctRNALeuGAG (1.1 s−1 µM−1 in Table 1). Similarly, MmLeuRS-CP1/LSD charged another double mutant (A6G + V-arm-5 nt) and MmtRNALeuUAA (another MmtRNALeu isoacceptor) with the same catalytic efficiency (0.78 s−1 µM−1) (Table 3). Interestingly, we found that MmtRNALeuUAA naturally exhibits a large loop of 5 nucleotides in the V-arm according to the genomic tRNA database.Figure 3.


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)

Mutations in MmtRNALeuUAG that impact editing activity. (A) Cloverleaf structure of MmtRNALeuUAG showing the mutations tested during the study. (B) AMP formation assay in the presence of 15 mM Nva catalyzed by 1 µM MmLeuRS-CP1/LSD in the presence of 5 µM wild-type MmtRNALeuUAG, C20U and C20U + V-arm-5 nt. (C) Graphical representations of AMP formation as a function of time. kobs values of AMP formation were calculated from the slopes, and these are shown in Table 4.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt185-F3: Mutations in MmtRNALeuUAG that impact editing activity. (A) Cloverleaf structure of MmtRNALeuUAG showing the mutations tested during the study. (B) AMP formation assay in the presence of 15 mM Nva catalyzed by 1 µM MmLeuRS-CP1/LSD in the presence of 5 µM wild-type MmtRNALeuUAG, C20U and C20U + V-arm-5 nt. (C) Graphical representations of AMP formation as a function of time. kobs values of AMP formation were calculated from the slopes, and these are shown in Table 4.
Mentions: To identify the structural determinants of tRNALeu responsible for LeuRS ability to discriminate tRNALeus from various species, we compared the tRNALeu sequences from E. coli and M. mobile and focused our attention on three differences between them: (i) the sixth base-pair in the acceptor stem of MmtRNALeuUAG is a wobble base pair (A6•••C67), whereas it is a Watson Crick base pair G6–C67 in EctRNALeuGAG; (ii) the loop of the V-arm of MmtRNALeuUAG contains three nucleotides; however, EctRNALeuGAG has a 4-nucleotide loop; (iii) nucleotide 20, located in the ‘variable pocket’ (20) of the D-loop, is always a U in EctRNALeus but always a C in MmtRNALeuUAG (Figure 3A). Therefore, a series of mutants of MmtRNALeuUAG was constructed. Firstly, the A6•••C67 pair was mutated to a Watson Crick base pair by introducing A6G or C67U mutations. Secondly, in the ‘variable pocket’, nucleotide C20 was changed to a U. Thirdly, the loop of the V-arm was enlarged from three nucleotides to four (V-arm-4 nt) or five (V-arm-5 nt), which are usual sizes for these loops in tRNALeus. MmLeuRS-CP1/LSD leucylated the C20U and V-arm-5 nt mutants at more than twice the catalytic efficiency of the wild-type MmtRNALeuUAG (from 0.3 to 0.75 and 0.70 s−1 µM−1, respectively). MmLeuRS-CP1/LSD leucylated the double mutant (C20U + V-arm-5 nt), where the C20U mutation and V-arm-5 nt mutation were present, and catalytic efficiency (kcat/Km 1.1 s−1 µM−1) (Table 3) almost reached the level of EctRNALeuGAG (1.1 s−1 µM−1 in Table 1). Similarly, MmLeuRS-CP1/LSD charged another double mutant (A6G + V-arm-5 nt) and MmtRNALeuUAA (another MmtRNALeu isoacceptor) with the same catalytic efficiency (0.78 s−1 µM−1) (Table 3). Interestingly, we found that MmtRNALeuUAA naturally exhibits a large loop of 5 nucleotides in the V-arm according to the genomic tRNA database.Figure 3.

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