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tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily.

Liu RJ, Long T, Zhou M, Zhou XL, Wang ED - Nucleic Acids Res. (2015)

Bottom Line: Our crystallographic study reveals that full-length EcTrmJ forms an unusual dimer in the asymmetric unit, with both the catalytic SPOUT domain and C-terminal extension forming separate dimeric associations.Based on these findings, we used electrophoretic mobility shift assay, isothermal titration calorimetry and enzymatic methods to identify amino acids within EcTrmJ that are involved in tRNA binding.We found that tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and that this process is regulated by the flexible hinge region that connects these two domains.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China University of Chinese Academy of Sciences, Beijing 100039, China.

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The tRNA substrates of TrmJ in Escherichia coli. (A) The cloverleaf structures of six E. coli tRNAs with a 2′-O-methylated ribose in position 32 from the MODOMICS database. (B and C) The methyltransferase activity of EcTrmJ for tRNA substrates. Error bars represent standard errors of three independent experiments.
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Figure 1: The tRNA substrates of TrmJ in Escherichia coli. (A) The cloverleaf structures of six E. coli tRNAs with a 2′-O-methylated ribose in position 32 from the MODOMICS database. (B and C) The methyltransferase activity of EcTrmJ for tRNA substrates. Error bars represent standard errors of three independent experiments.

Mentions: A total of six tRNAs from E. coli have 2′-O-methylated nucleosides at position 32, as presented in the MODOMICS database (1). They are tRNAGln1(UUG), tRNAGln2(CUG), tRNASer1(UGA), tRNAfMet1(CAU), tRNAfMet2(CAU), and tRNATrp1(CCA) (Figure 1A). In this present study, we transcribed all six candidate tRNAs in vitro and measured their methylation catalyzed by EcTrmJ. Furthermore, tRNAGln2 and tRNASer1 had been previously identified as substrates of EcTrmJ (38,39), and the other four candidates could also be methylated by EcTrmJ (Figure 1B and C). Therefore, all of these six tRNAs are substrates of EcTrmJ. Subsequently, we measured the steady-state kinetic parameters of EcTrmJ for these six substrates (Table 1). According to the kinetic parameters of methylation, the tRNA substrates could be divided into two groups as follows: Group 1 with tRNAfMet1, tRNAfMet2, and tRNATrp1 where Km values were all below 1 μM and kcat values ranged from 1.31 to 1.52 min−1, and Group 2 with tRNAGln1, tRNAGln2, and tRNASer1, characterized by higher Km values >5 μM and kcat values that were similar to those of Group 1 (Table 1). Collectively, our data show that all six EctRNAs with a methylated 2′-O-ribose at position 32 are substrates of EcTrmJ. In the following experiments, we chose to study tRNAfMet1, which has a lower Km value, to characterize tRNA recognition by EcTrmJ.


tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily.

Liu RJ, Long T, Zhou M, Zhou XL, Wang ED - Nucleic Acids Res. (2015)

The tRNA substrates of TrmJ in Escherichia coli. (A) The cloverleaf structures of six E. coli tRNAs with a 2′-O-methylated ribose in position 32 from the MODOMICS database. (B and C) The methyltransferase activity of EcTrmJ for tRNA substrates. Error bars represent standard errors of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4551947&req=5

Figure 1: The tRNA substrates of TrmJ in Escherichia coli. (A) The cloverleaf structures of six E. coli tRNAs with a 2′-O-methylated ribose in position 32 from the MODOMICS database. (B and C) The methyltransferase activity of EcTrmJ for tRNA substrates. Error bars represent standard errors of three independent experiments.
Mentions: A total of six tRNAs from E. coli have 2′-O-methylated nucleosides at position 32, as presented in the MODOMICS database (1). They are tRNAGln1(UUG), tRNAGln2(CUG), tRNASer1(UGA), tRNAfMet1(CAU), tRNAfMet2(CAU), and tRNATrp1(CCA) (Figure 1A). In this present study, we transcribed all six candidate tRNAs in vitro and measured their methylation catalyzed by EcTrmJ. Furthermore, tRNAGln2 and tRNASer1 had been previously identified as substrates of EcTrmJ (38,39), and the other four candidates could also be methylated by EcTrmJ (Figure 1B and C). Therefore, all of these six tRNAs are substrates of EcTrmJ. Subsequently, we measured the steady-state kinetic parameters of EcTrmJ for these six substrates (Table 1). According to the kinetic parameters of methylation, the tRNA substrates could be divided into two groups as follows: Group 1 with tRNAfMet1, tRNAfMet2, and tRNATrp1 where Km values were all below 1 μM and kcat values ranged from 1.31 to 1.52 min−1, and Group 2 with tRNAGln1, tRNAGln2, and tRNASer1, characterized by higher Km values >5 μM and kcat values that were similar to those of Group 1 (Table 1). Collectively, our data show that all six EctRNAs with a methylated 2′-O-ribose at position 32 are substrates of EcTrmJ. In the following experiments, we chose to study tRNAfMet1, which has a lower Km value, to characterize tRNA recognition by EcTrmJ.

Bottom Line: Our crystallographic study reveals that full-length EcTrmJ forms an unusual dimer in the asymmetric unit, with both the catalytic SPOUT domain and C-terminal extension forming separate dimeric associations.Based on these findings, we used electrophoretic mobility shift assay, isothermal titration calorimetry and enzymatic methods to identify amino acids within EcTrmJ that are involved in tRNA binding.We found that tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and that this process is regulated by the flexible hinge region that connects these two domains.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China University of Chinese Academy of Sciences, Beijing 100039, China.

Show MeSH
Related in: MedlinePlus