Structural Studies of Selenocysteine Synthetase (SELA) from Escherichia coli
Cassago, A1; Oliveira, CL2; Torriani, IL2 e Thiemann, OH1
1Laboratório de Cristalografia de Proteínas e Biologia Estrutural Centro de Biotecnologia Molecular Estrutural – CEPID. Instituto de Física de São Carlos, Universidade de São Paulo, SP, Brazil.
2Laboratório Nacional de Luz Síncrotron, Campinas, SP, Brazil.
casslex@if.sc.usp.br
The biosynthesis of the 21th amino acid, selenocysteine (Sec or U) requires a complex enzymatic machinery composed of Selenocysteine Synthase (SELA) exclusive of prokariotes, Selenocysteine Elongation Factor (SELB or EFSec), Selenophosphate Synthetase (SELD) and a specific tRNAsec named Selenocysteine Insertion tRNA (SELC). Our aim is to elucidate the protein-tRNA recognition mechanism by the structural investigation of Escherichia coli SELA in solution by Small Angle X-ray Scattering (SAXS) with and without tRNA.
SAXS measurements of the SELA protein in the absence of the tRNASec , were performed using the SAXS1 beamline at the Laboratório Nacional de Luz Síncrotron (LNLS). Data analysis allowed the determination of the global SELA structure as a homodecamer composed of five SELA dimers with a maximum diameter of 185Å, a molecular mass of 527 +/- 27kDa and a radius of gyration of 67.3Å. Higher resolution data were subsequently collected for SELA in the presence and absence of tRNAsec using 2D detection at the SAXS2 beamline of the LNLS. Clear differences were noted between the scattering curves and pair distance distribution functions calculated for the two cases indicated the formation of complexes between the protein SELA and tRNASec.
Since these preliminary results are consistent those obtained previously by Transmission Electron Microscopy (TEM), real space information will be used as a guide to perform shape analysis from the SAXS data, to obtain the solution structure of SELA and obtain evidence of its binding with tRNAsec .
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