Structural studies of the native Seryl-tRNA Synthetase and in interaction with cognates tRNAs from Trypanosoma brucei

Author(s)

Daiana Evelin Martil

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2014-04-17

Examining board members

Otavio Henrique Thiemann; José Ramon Beltran Abrego; Yvonne Primerano Mascarenhas; Alessandro Silva Nascimento; Luis Paulo Barbour Scott

• Advisor: Otavio Henrique Thiemann

Abstract

The synthesis of selenocysteine and its co-translational incorporation in selenoproteins in response to a UGA codon in frame require complex molecular machinery. In eukaryotes, components that participate in the reaction of selenocysteine formation were identified: SeryltRNA synthetase (SerRS), O-phosphoseryl-tRNA kinase (PSTK), SECIS Binding Protein 2 - SBP2, a selenocysteine-specific elongation factor (EFSec), selenophosphate synthetase 1 (SPS1) and selenophosphate synthetase 2 (SPS2), SEPSECS, SECp43 RNA binding protein, ribosomal protein L30, selenocysteine tRNA (tRNASec, SELC), and a specific sequence in the messenger RNA (SECIS element). The first step for selenocysteine incorporating is performed by SerRS that aminoacylates the tRNA with serine through serine activation by Mg2+ and ATP leading to the formation of an intermediate linked to the enzyme (Ser-AMP). Subsequently, the change of the Ser radical to tRNASec takes place followed by the enzymatic conversion of Ser-tRNASec to Sec-tRNASec. Through in silico analysis our group has identified components of the selenocysteine insertion machinery in species of Kinetoplastida. Homologues of tRNASec and the enzymes TbSerRS, TbSPS2, TbPSTK, TbSepSecS and TbEFSec were identified. Our main target is the structural study of the native SerRS from Trypanosoma brucei and SerRS in complex with the tRNASec and the tRNASer isoforms. A new methodology in the purification process of this enzyme has been developed, and through molecular exclusion chromatography, dynamic light scattering and analytical ultracentrifugation techniques we were able to determine the oligomeric state of TbSerRS. The result of dimers in solution corroborated with the data reported in the literature. Moreover, we were able to verify through studies of enzyme kinetics that the enzyme is active. The sedimentation equilibrium analytical ultracentrifugation technique also demonstrated the formation of the SerRS-tRNA complex, however, it did not allow the definition of the complex stoichiometry. Structural studies of the native enzyme and its interaction with SELC, tRNASer isoforms, L-serine, a non-hydrolyzable AMP analog, MgCl2, and smaller portions of tRNAs were performed by X-ray diffraction crystallography. Through this technique, seventeen data sets were collected, processed, and are being submitted to refinement processes. Initial structural analysis allowed the confirmation of the presence of two glycerol molecules in each monomer in the active site region in the native structure of TbSerRS and one dAMP molecule in the TbSerRS-dAMP complex. (AU)

• FAPESP's process: 07/06590-3 - Molecular characterization of the selenocisteine synthesis pathway: structural and functional investigation of the human phosphoseril tRNA kinase (PSTK)
• Grantee: Daiana Evelin Martil
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)