Plant-exclusive domain of trans-editing enzyme ProXp-ala confers dimerization and enhanced tRNA binding.
J Biol Chem
; 298(9): 102255, 2022 09.
Article
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| MEDLINE
| ID: mdl-35835222
ABSTRACT
Faithful translation of the genetic code is critical for the viability of all living organisms. The trans-editing enzyme ProXp-ala prevents Pro to Ala mutations during translation by hydrolyzing misacylated Ala-tRNAPro that has been synthesized by prolyl-tRNA synthetase. Plant ProXp-ala sequences contain a conserved C-terminal domain (CTD) that is absent in other organisms; the origin, structure, and function of this extra domain are unknown. To characterize the plant-specific CTD, we performed bioinformatics and computational analyses that provided a model consistent with a conserved α-helical structure. We also expressed and purified wildtype Arabidopsis thaliana (At) ProXp-ala in Escherichia coli, as well as variants lacking the CTD or containing only the CTD. Circular dichroism spectroscopy confirmed a loss of α-helical signal intensity upon CTD truncation. Size-exclusion chromatography with multiangle laser-light scattering revealed that wildtype At ProXp-ala was primarily dimeric and CTD truncation abolished dimerization in vitro. Furthermore, bimolecular fluorescence complementation assays in At protoplasts support a role for the CTD in homodimerization in vivo. The deacylation rate of Ala-tRNAPro by At ProXp-ala was also significantly reduced in the absence of the CTD, and kinetic assays indicated that the reduction in activity is primarily due to a tRNA binding defect. Overall, these results broaden our understanding of eukaryotic translational fidelity in the plant kingdom. Our study reveals that the plant-specific CTD plays a significant role in substrate binding and canonical editing function. Through its ability to facilitate protein-protein interactions, we propose the CTD may also provide expanded functional potential for trans-editing enzymes in plants.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas de Plantas
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Biosíntesis de Proteínas
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ARN de Transferencia
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Prolina
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Arabidopsis
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Alanina
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Multimerización de Proteína
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Aminoacil-ARNt Sintetasas
Idioma:
En
Revista:
J Biol Chem
Año:
2022
Tipo del documento:
Article
País de afiliación:
Estados Unidos