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| Author(s): |
Livia Regina Manzine
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Carlos. |
| Institution: | Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT) |
| Defense date: | 2012-01-25 |
| Examining board members: |
Otavio Henrique Thiemann;
Angela Kaysel Cruz;
José Ribamar dos Santos Ferreira Júnior;
Alessandro Silva Nascimento;
Marcos Vicente de Albuquerque Salles Navarro
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| Advisor: | Otavio Henrique Thiemann |
| Abstract | |
The formation and incorporation of the amino acid selenocysteine in Escherichia coli is an event directed by cotraducional UGA codon and involves a complex biosynthesis pathway whose main proteins are: Selenocysteine synthase (SELA), elongation factor of selenocysteine (SELB), Selenophosphate synthetase (SELD), Seryl-tRNA synthetase, a selenocysteine tRNA (tRNAsec or SELC) and a specific sequence on the messenger RNA, called Selenocysteine insertion sequence (SECIS). The incorporation of selenocysteine in proteins of bacteria begins with the tRNAsec aminoacylation with serine by the enzyme Seryl-tRNA synthetase resulting in seryl-tRNAsec which is subsequently converted to selenocysteyl-tRNAsec by the enzyme Selenocysteine synthase (SELA). The selenium used in the conversion reaction is provided by Selenophosphate synthetase as selenophosphate and finally, the selenocysteyl-tRNAsec is delivered by the factor SELB to the ribosome. The present study focused on biochemical and biophysical studies of SELA protein and analysis of its interaction with the specific ligand (SELC) for determination of binding parameters involved in the formation of the complex. The gene coding for SELA protein was subcloned, expressed in WL81460(DE3) bacterial strain and the protein was purified as described in the literature; however a new, faster and more efficient method for its purification was developed. Studies of gel filtration, native gel electrophoresis, isoelectric focusing, circular dichroism, intrinsic fluorescence spectroscopy and chemical crosslinking provided a better characterization of SELA protein and a greater understanding of its behavior in solution. Analysis of fluorescence anisotropy spectroscopy revealed that SELA was able to associate in a supramolecular state. This analysis was mainly corroborated by data from electron microscopy employing negative staining technique. Fluorescence anisotropy methodology allowed us to analyse the interaction of SELA protein with the specific ligand SELC, as well as with others mutated tRNAs enabling a mapping of important regions in SELC for interaction. In addition, fluorescence anisotropy technique was also successfully used in determining the stoichiometry ratio of the complex SELA-SELC, showing a proportion of 1 molecule of SELA to 10 tRNAs, contraring to the literary data published in 1991 and 1992. (AU) | |