人线粒体亮氨酸tRNA结构和功能的研究超嗜热菌亮氨酰-tRNA合成酶双亚基的体外重组
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摘要
线粒体性脑肌病,乳酸中毒和中风样发作综合症(MELAS症)是一种罕见的先天性线粒体DNA病,有报道指出,位于编码人线粒体tRNA~(Leu)(UUR)基因中的五种点突变与MELAS症相关。我们构建并转录获得了这些tRNA突变体,通过体外的氨基酰化实验,测定了这些点突变对tRNA接受茎活力的影响。结果显示,和野生型tRNA相比,带有这五种点突变的tRNA~(Leu)(UUR)转录子的氨基酰化水平都有不同程度的降低。进一步的热稳定性实验证明,氨基酰化水平最低的A3243G突变体和T3291C突变体的结构不稳定。另外,T3291C突变体能够竞争性地抑制野生型tRNA的亮氨酰化,暗示携带该突变的tRNA可能作为一种抑制剂存在于患者的线粒体内。
人线粒体亮氨酸tRNA的CUN等受体(hmtRNA~(Leu)(CUN))负责解码线粒体蛋白质编码系统中使用频率最高的密码子,目前有六个致病性点突变被定位于它的基因内部。本文的体外实验显示,和野生型的hmtRNA~(Leu)(CUN)转录子
The mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-likeepisodes syndrome (MELAS) is a rare congenital disorder of mitochondrial DNA(mtDNA). Five single nucleotide substitutions within the human mitochondrialtRNA~(Leu)(UUR) gene have been reported to be associated with MELAS. Here weprovide in vitro evidence that the aminoacylation capacities of these fivehmtRNALeu(UUR) transcripts are reduced to different extents relative to thewild-type hmtRNA~(Leu)(UUR) transcript. A thermal denaturation experiment showedthat the A3243G and T3291C mutants, which were the least charged by LeuRS, havefragile structures. In addition, the T3291C mutant can inhibit aminoacylation of thewild-type hmtRNA~(Leu)(UUR), indicating that it may act as an inhibitor in themitochondrial heteroplasmic environment.
The human mitochondrial tRNALeu(CUN) (hmtRNALeu(CUN)) corresponds tothe most abundant codon for leucine in human mitochondrial protein genes. Here, invitro studies reveal that the U48C substitution in hmtRNALeu(CUN), whichcorresponds to the pathological T12311C gene mutation, improved theaminoacylation efficiency of hmtRNALeu(CUN). Enzymatic probing suggested amore flexible secondary structure in the wild type hmtRNALeu(CUN) transcriptcompared to the U48C mutant. Structural analysis revealed that the flexibility ofhmtRNALeu(CUN) facilitates a T-stem slip resulting in 2 potential tertiary structures.Several rationally designed tRNALeu(CUN) mutants were generated to examine thestructural and functional consequences of the T-stem slip. Examination of thesehmtRNALeu(CUN) mutants indicated that the T-stem slip could change the tRNAtertiary structure as well as govern its charging activity. These results suggest anovel, self-regulation mechanism of tRNA structure and function. Different tRNAtranscripts were bound on beads respectively, by which we screened tRNA bindingprotein from human mitochondria. Using this method, we found an unknown proteinthat can recognize specific tRNA structure. This result cast a new light on themechanism of related mitochondrial disease.
The Aquifex aeolicus αβ-LeuRS is the only known heterodimericLeucyl-tRNA synthetase. The His6-tagged α-subunit gene was cloned into anexpression vector, which was transferred into E. coli BL21(DE3). After lowtemperature induced expression, the soluble His-α protein was purified in a singlestep by metal (Ni2+) chelate affinity chromatography. We mixed the His-α and β invitro and found that the recombinant protein recovered leucylation activity.Compared with several other factors, urea promotes recombinant efficiencyobviously.
人线粒体亮氨酸tRNA的CUN等受体(hmtRNA~(Leu)(CUN))负责解码线粒体蛋白质编码系统中使用频率最高的密码子,目前有六个致病性点突变被定位于它的基因内部。本文的体外实验显示,和野生型的hmtRNA~(Leu)(CUN)转录子
The mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-likeepisodes syndrome (MELAS) is a rare congenital disorder of mitochondrial DNA(mtDNA). Five single nucleotide substitutions within the human mitochondrialtRNA~(Leu)(UUR) gene have been reported to be associated with MELAS. Here weprovide in vitro evidence that the aminoacylation capacities of these fivehmtRNALeu(UUR) transcripts are reduced to different extents relative to thewild-type hmtRNA~(Leu)(UUR) transcript. A thermal denaturation experiment showedthat the A3243G and T3291C mutants, which were the least charged by LeuRS, havefragile structures. In addition, the T3291C mutant can inhibit aminoacylation of thewild-type hmtRNA~(Leu)(UUR), indicating that it may act as an inhibitor in themitochondrial heteroplasmic environment.
The human mitochondrial tRNALeu(CUN) (hmtRNALeu(CUN)) corresponds tothe most abundant codon for leucine in human mitochondrial protein genes. Here, invitro studies reveal that the U48C substitution in hmtRNALeu(CUN), whichcorresponds to the pathological T12311C gene mutation, improved theaminoacylation efficiency of hmtRNALeu(CUN). Enzymatic probing suggested amore flexible secondary structure in the wild type hmtRNALeu(CUN) transcriptcompared to the U48C mutant. Structural analysis revealed that the flexibility ofhmtRNALeu(CUN) facilitates a T-stem slip resulting in 2 potential tertiary structures.Several rationally designed tRNALeu(CUN) mutants were generated to examine thestructural and functional consequences of the T-stem slip. Examination of thesehmtRNALeu(CUN) mutants indicated that the T-stem slip could change the tRNAtertiary structure as well as govern its charging activity. These results suggest anovel, self-regulation mechanism of tRNA structure and function. Different tRNAtranscripts were bound on beads respectively, by which we screened tRNA bindingprotein from human mitochondria. Using this method, we found an unknown proteinthat can recognize specific tRNA structure. This result cast a new light on themechanism of related mitochondrial disease.
The Aquifex aeolicus αβ-LeuRS is the only known heterodimericLeucyl-tRNA synthetase. The His6-tagged α-subunit gene was cloned into anexpression vector, which was transferred into E. coli BL21(DE3). After lowtemperature induced expression, the soluble His-α protein was purified in a singlestep by metal (Ni2+) chelate affinity chromatography. We mixed the His-α and β invitro and found that the recombinant protein recovered leucylation activity.Compared with several other factors, urea promotes recombinant efficiencyobviously.
引文
1. Woese, C. R., Olsen, G. J., Ibba, M., and S?ll, D. (2000) Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process. Microbiol. Mol. Biol. Rev., 64, 202-236.
2. Fersht, A.R. (1985) Enzyme Structure and Mechanism, Second Edition (New York: W. H. Freeman and Company), 354–358.
3. Giegé, R., Sissler, M. and Florentz, C. (1998) Universal rules and idiosyncratic features in tRNA identity. Nucleic Acid Res., 26, 5017–5035.
4. Saks, M.E., Sampson, J.R. and Abelson, J. (1994) The transfer RNA identity problem: a search for rules. Science, 263, 191–197.
5. Anderson, S. et al. (1981) Sequence and organization of the human mitochondrial genome. Nature, 290, 457-465.
6. Sprinzl, M., and Vassilenko, K.S. (2005) Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res., 33, 139-140.
7. Soll, D. (1993) Transfer RNA: an RNA for all season. In Gestelandm, R.F. and Atkins, J.F. (eds.), The RNA World, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY pp 157-183.
8. Helm, M., Brule, H., Friede, D., Giegé, R., Putz, D., and Florentz, C. (2000) Search for characteristic structural features of mammalian mitochondrial tRNAs. RNA, 6, 1356-1379.
9. Pesole, G., Gissi, C., DeChirico, A., and Saccone, C. (1999) Nucleotide substitution rate of mammalian mitochondrial genomes. J. Mol. Evol., 48, 427–434.
10. Wittenhagen, L.M., and Kelley, S.O. (2003) Impact of disease-related mitochondrial mutations on tRNA structure and function. Trends Biochem. Sci., 28, 605-611.
11. Piechota, J., Mroczek, K., and Bartnik, E. (2001) MELAS as an example of a mitochondrial disease. J. Appl. Genet., 42, 351-358.
12. Goto, Y., Nonaka, I., and Horai, S. (1990) A mutation in the tRNALeu(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies. Nature, 348, 651–653.
13. Morten, K. J., Cooper, J. M., Brown, G. K., Lake, B. D., Pike, D., and Poulton, J. (1993) A new point mutation associated with mitochondrial encephalomyopathy. Hum. Mol. Genet., 2, 2081-2087.
14. Sato, W., Hayasaka, K., Shoji, Y., Takahashi, T., Takada, G., Saito, M., Fukawa, O., and Wachi, E. (1994) A mitochondrial tRNALeu(UUR) mutation at 3,256 associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Biochem. Mol. Biol. Int., 33, 1055-1061.
15. Goto, Y., Nonaka, I., and Horai, S. (1991) A new mtDNA mutation associatedwith mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-likeepisodes (MELAS). Biochim. Biophys. Acta, 1097, 238–240.
16. Goto, Y., Tsugane, K., Tanabe, Y., Nnaka, I., and Horai, S. (1994) A new pointmutation at nucleotide pair 3291 of the mitochondrial tRNALeu(UUR) gene in apatient with mitochondrial myopathy, encephalopathy, lactic acidosis, andstroke-like episodes (MELAS). Biochem. Biophys. Res. Commun., 202,1624-1630.
17. Chomyn, A., Enriquez, J. A., Micol, V., Fernandez-Silva, P., and Attardi, G.(2000) The mitochondrial myopathy, encephalopathy, lactic acidosis, andstroke-like episode syndrome-associated human mitochondrial tRNALeu(UUR)mutation causes aminoacylation deficiency and concomitant reduced associationof mRNA with ribosomes. J. Biol. Chem., 275, 19198-19209.
18. Park, H., Davidson, E., and King, M. P. (2003) The pathogenic A3243Gmutation in human mitochondrial tRNALeu(UUR) decreases the efficiency ofaminoacylation. Biochemistry, 42, 958-964.
19. Sohm, B., Frugier, M., Brule, H., Olszak, K., Przykorska, A., and Florentz, C.(2003) Towards understanding human mitochondrial leucine aminoacylationidentity. J. Mol. Biol., 328, 995-1010.
20. Enriquez, J. A., Cabzas-Herrera, J., Bayona-Bafaluy, M. P., and Attardi, G. (2000) Very rare complementation between mitochondria carrying different mitochondrial DNA mutations points to intrinsic genetic autonomy of the organelles in cultured human cells. J. Biol. Chem., 275, 11207-11215.
21. Takai, D., Isobe, K., and Hayashi, J. (1999) Transcomplementation between different types of respiration-deficient mitochondria with different pathogenic mutant mitochondrial DNAs. J. Biol. Chem., 274, 11199-11202.
22. Li, Y., Wang, E.D., and Wang, Y.L. (1999) A modified procedure for fast purification of T7 RNA polymerase. Protein Expr. Purif., 16, 355-388.
23. Yao, Y.N., Wang, L., Wu, X.F., and Wang, E.D. (2003) Human mitochondrial leucyl-tRNA synthetase with high activity produced from Escherichia coli. Protein Expr. Purif., 30, 112-116.
24. Puglisi, J.D., and Tinoco, I.Jr. (1989) Absorbance melting curves of RNA. Methods Enzymol., 180, 304–325
25. Dunn, J.J., and Studier, F.W. (1983) Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol., 166. 477-535.
26. Fechter, P., Rudinger, J., Giegé, R., and Theobald-Dietrich, A. (1998) Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity. FEBS Lett., 436, 99-103.
27. Jacobs, H.T., and Holt, I.J. (2000) The np 3243 MELAS mutation: damned if you aminoacylate, damned if you don't. Hum. Mol. Genet., 9, 463-465.
28. Kelley, S.O., Steinberg, S.V., and Schimmel, P. (2000) Functional defects of pathogenic human mitochondrial tRNAs related to structural fragility. Nat. Struct. Biol., 7, 862-865.
29. Kelley, S.O., Steinberg, S.V., and Schimmel, P. (2001) Fragile T-stem in disease-associated human mitochondrial tRNA sensitizes structure to local and distant mutations. J. Biol. Chem., 276, 10607-10611.
30. Sissler, M., Helm, M., Frugier, M., Giegé, R., and Florentz, C. (2004) Aminoacylation properties of pathology-related human mitochondrial tRNALys variants. RNA, 10, 841-853.
31. Wittenhagen, L.M., and Kelley, S.O. (2002) Dimerization of a pathogenic human mitochondrial tRNA. Nat. Struct. Biol., 9, 586-590.
32. Feuermann, M., Francisci, S., Rinaldi, T., De Luca, C., Rohou, H., Frontali, L., and Bolotin-Fukuhara, M. (2003) The yeast counterparts of human 'MELAS' mutations cause mitochondrial dysfunction that can be rescued by overexpression of the mitochondrial translation factor EF-Tu. EMBO Rep., 4, 53-58.
33. Quigley, G.J., and Rich, A. (1976) Structural domains of transfer RNA molecules. Science, 194, 796-806.
34. Sohm, B., Sissler, M., Park, H., King, M.P., and Florentz, C. (2004) Recognition of human mitochondrial tRNALeu(UUR) by its cognate leucyl-tRNA synthetase. J. Mol. Biol., 339, 17-29.
35. Uziel, G., Carrara, F., Granata, T., Lamantea, E., Mora, M., and Zeviani, M. (2000) Neuromuscular syndrome associated with the 3291TC mutation of mitochondrial DNA: a second case. Neuromuscul. Disord., 10, 415-418.
36. Roy M.D., Wittenhagen L.M., Vozzella B.E., and Kelley S.O. (2004) Interdomain communication between weak structural elements within a disease-related human tRNA. Biochemistry, 43 384-392.
2. Fersht, A.R. (1985) Enzyme Structure and Mechanism, Second Edition (New York: W. H. Freeman and Company), 354–358.
3. Giegé, R., Sissler, M. and Florentz, C. (1998) Universal rules and idiosyncratic features in tRNA identity. Nucleic Acid Res., 26, 5017–5035.
4. Saks, M.E., Sampson, J.R. and Abelson, J. (1994) The transfer RNA identity problem: a search for rules. Science, 263, 191–197.
5. Anderson, S. et al. (1981) Sequence and organization of the human mitochondrial genome. Nature, 290, 457-465.
6. Sprinzl, M., and Vassilenko, K.S. (2005) Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res., 33, 139-140.
7. Soll, D. (1993) Transfer RNA: an RNA for all season. In Gestelandm, R.F. and Atkins, J.F. (eds.), The RNA World, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY pp 157-183.
8. Helm, M., Brule, H., Friede, D., Giegé, R., Putz, D., and Florentz, C. (2000) Search for characteristic structural features of mammalian mitochondrial tRNAs. RNA, 6, 1356-1379.
9. Pesole, G., Gissi, C., DeChirico, A., and Saccone, C. (1999) Nucleotide substitution rate of mammalian mitochondrial genomes. J. Mol. Evol., 48, 427–434.
10. Wittenhagen, L.M., and Kelley, S.O. (2003) Impact of disease-related mitochondrial mutations on tRNA structure and function. Trends Biochem. Sci., 28, 605-611.
11. Piechota, J., Mroczek, K., and Bartnik, E. (2001) MELAS as an example of a mitochondrial disease. J. Appl. Genet., 42, 351-358.
12. Goto, Y., Nonaka, I., and Horai, S. (1990) A mutation in the tRNALeu(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies. Nature, 348, 651–653.
13. Morten, K. J., Cooper, J. M., Brown, G. K., Lake, B. D., Pike, D., and Poulton, J. (1993) A new point mutation associated with mitochondrial encephalomyopathy. Hum. Mol. Genet., 2, 2081-2087.
14. Sato, W., Hayasaka, K., Shoji, Y., Takahashi, T., Takada, G., Saito, M., Fukawa, O., and Wachi, E. (1994) A mitochondrial tRNALeu(UUR) mutation at 3,256 associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Biochem. Mol. Biol. Int., 33, 1055-1061.
15. Goto, Y., Nonaka, I., and Horai, S. (1991) A new mtDNA mutation associatedwith mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-likeepisodes (MELAS). Biochim. Biophys. Acta, 1097, 238–240.
16. Goto, Y., Tsugane, K., Tanabe, Y., Nnaka, I., and Horai, S. (1994) A new pointmutation at nucleotide pair 3291 of the mitochondrial tRNALeu(UUR) gene in apatient with mitochondrial myopathy, encephalopathy, lactic acidosis, andstroke-like episodes (MELAS). Biochem. Biophys. Res. Commun., 202,1624-1630.
17. Chomyn, A., Enriquez, J. A., Micol, V., Fernandez-Silva, P., and Attardi, G.(2000) The mitochondrial myopathy, encephalopathy, lactic acidosis, andstroke-like episode syndrome-associated human mitochondrial tRNALeu(UUR)mutation causes aminoacylation deficiency and concomitant reduced associationof mRNA with ribosomes. J. Biol. Chem., 275, 19198-19209.
18. Park, H., Davidson, E., and King, M. P. (2003) The pathogenic A3243Gmutation in human mitochondrial tRNALeu(UUR) decreases the efficiency ofaminoacylation. Biochemistry, 42, 958-964.
19. Sohm, B., Frugier, M., Brule, H., Olszak, K., Przykorska, A., and Florentz, C.(2003) Towards understanding human mitochondrial leucine aminoacylationidentity. J. Mol. Biol., 328, 995-1010.
20. Enriquez, J. A., Cabzas-Herrera, J., Bayona-Bafaluy, M. P., and Attardi, G. (2000) Very rare complementation between mitochondria carrying different mitochondrial DNA mutations points to intrinsic genetic autonomy of the organelles in cultured human cells. J. Biol. Chem., 275, 11207-11215.
21. Takai, D., Isobe, K., and Hayashi, J. (1999) Transcomplementation between different types of respiration-deficient mitochondria with different pathogenic mutant mitochondrial DNAs. J. Biol. Chem., 274, 11199-11202.
22. Li, Y., Wang, E.D., and Wang, Y.L. (1999) A modified procedure for fast purification of T7 RNA polymerase. Protein Expr. Purif., 16, 355-388.
23. Yao, Y.N., Wang, L., Wu, X.F., and Wang, E.D. (2003) Human mitochondrial leucyl-tRNA synthetase with high activity produced from Escherichia coli. Protein Expr. Purif., 30, 112-116.
24. Puglisi, J.D., and Tinoco, I.Jr. (1989) Absorbance melting curves of RNA. Methods Enzymol., 180, 304–325
25. Dunn, J.J., and Studier, F.W. (1983) Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol., 166. 477-535.
26. Fechter, P., Rudinger, J., Giegé, R., and Theobald-Dietrich, A. (1998) Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity. FEBS Lett., 436, 99-103.
27. Jacobs, H.T., and Holt, I.J. (2000) The np 3243 MELAS mutation: damned if you aminoacylate, damned if you don't. Hum. Mol. Genet., 9, 463-465.
28. Kelley, S.O., Steinberg, S.V., and Schimmel, P. (2000) Functional defects of pathogenic human mitochondrial tRNAs related to structural fragility. Nat. Struct. Biol., 7, 862-865.
29. Kelley, S.O., Steinberg, S.V., and Schimmel, P. (2001) Fragile T-stem in disease-associated human mitochondrial tRNA sensitizes structure to local and distant mutations. J. Biol. Chem., 276, 10607-10611.
30. Sissler, M., Helm, M., Frugier, M., Giegé, R., and Florentz, C. (2004) Aminoacylation properties of pathology-related human mitochondrial tRNALys variants. RNA, 10, 841-853.
31. Wittenhagen, L.M., and Kelley, S.O. (2002) Dimerization of a pathogenic human mitochondrial tRNA. Nat. Struct. Biol., 9, 586-590.
32. Feuermann, M., Francisci, S., Rinaldi, T., De Luca, C., Rohou, H., Frontali, L., and Bolotin-Fukuhara, M. (2003) The yeast counterparts of human 'MELAS' mutations cause mitochondrial dysfunction that can be rescued by overexpression of the mitochondrial translation factor EF-Tu. EMBO Rep., 4, 53-58.
33. Quigley, G.J., and Rich, A. (1976) Structural domains of transfer RNA molecules. Science, 194, 796-806.
34. Sohm, B., Sissler, M., Park, H., King, M.P., and Florentz, C. (2004) Recognition of human mitochondrial tRNALeu(UUR) by its cognate leucyl-tRNA synthetase. J. Mol. Biol., 339, 17-29.
35. Uziel, G., Carrara, F., Granata, T., Lamantea, E., Mora, M., and Zeviani, M. (2000) Neuromuscular syndrome associated with the 3291TC mutation of mitochondrial DNA: a second case. Neuromuscul. Disord., 10, 415-418.
36. Roy M.D., Wittenhagen L.M., Vozzella B.E., and Kelley S.O. (2004) Interdomain communication between weak structural elements within a disease-related human tRNA. Biochemistry, 43 384-392.