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| Author(s): |
Jéssica Fernandes Scortecci
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: | 2019-02-04 |
| Examining board members: |
Otavio Henrique Thiemann;
Rosangela Itri;
João Renato Carvalho Muniz;
Carla Ribeiro Polycarpo;
Carlos Henrique Inacio Ramos
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| Advisor: | Otavio Henrique Thiemann |
| Abstract | |
The existence of co-translationally encoded amino acids by the genetic code has stimulated studies on the mechanisms of synthesis, recognition, and incorporation into new polypeptide chains. As an example, the selenocysteine (Sec) biosynthesis pathway, present in eukaryotes and prokaryotes, where the amino acid incorporation occurs at the canonical UGA stop-codon. In Bacteria, the Sec biosynthesis pathway is formed by Selenocysteine synthase (SelA), Specific Elongation Factor (SelB), Selenophosphate synthetase (SelD), Seryl-tRNA synthetase (SerRS) and Selenocysteine lyase (CsdB). The synthesis route also needs two RNAs; a specific tRNA (tRNASec) and a sequence in the mRNA (SelenoCysteine Insertion Sequence - SECIS) that encodes for the in-frame UGA Sec incorporation. In eukaryotes, the pathway is distinguished through the presence of O-phosphoseryl-tRNASec kinase (PSTK) and Selenocysteinyl-tRNASec synthase (SepSecS), replacing SelA, also the presence of SECIS binding proteins (SBP2). Once selenium presents high cell toxicity, it is crucial to fully understand the catalytic metabolism and complex formation for the tRNASec incorporation. In 2009, CsdB and SelD interaction was proposed, however, it has not been experimentally demonstrated until now. Thus, this project reports at the first time the biophysical and structural characterization of bacterial CsdB and SelD macromolecular interaction, indicating to a high-affinity interaction between these enzymes for the complex formation. Biophysical assays showed that the complex increased the thermal stability and structural studies showed a low-resolution model also indicating the macromolecule asymmetry. In addition, our research group reported in 2013 the putative SBP2 sequence in N. gruberi, the non-pathogenic amoeba used as a model for studies of N. fowleri, known as human infective, responsible for the pathology known as the Primary Amebic Meningoencephalitis. Moreover, this project also reports, at the first time, the experimental presence of N. gruberi SBP2. The SBP2.SECIS was also characterized by several biophysical methods. NgSBP2 has a high percentage of regions of disorder and access to each element SECIS presents due to interaction. Thus, this study was promoted in advance on the molecular interactions present in the incorporation of selenocysteines, being important for the understanding of the molecular determinants of the interaction between protein-protein and RNA-protein. (AU) | |
| FAPESP's process: | 16/20977-7 - Study of the selenocysteine incorporation pathway: understanding the macromolecular interaction mechanism |
| Grantee: | Jessica Fernandes Scortecci |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |