STUDI EKSPRESI MUTAN SUP45 ALLOSUPPRESSOR DAN SENSITIF TEMPERATUR PADA RAGI SACCHAROMYCES CEREVISIAE

• Main Author: NENGAH WIRAJANA, I
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/1737
• Institution: Institut Teknologi Bandung

Mechanism of translational termination in eukaryote system has not been understood in detail yet. This process is mediated by two release factors, eRF1 and eRF3, encoded by the SUP45 and SUP35 genes respectively. To understand the mechanism of translational termination, particularly the role of eRFI protein in yeast Saccharomyces cerevisiae, the sup45 mutants were constructed. Phenotypic of the mutants including allosuppressor, temperature sensitivity and paramomycine hypersensitivity were characterized. The phenotypic of allosuppressor might be due to lower cellular levels of eRFI or by amino acid substitution of eRFI in the mutant. In this research, expression of eRFI in the sup45 mutants exhibiting allosuppressor and temperature sensitivity phenotypes had been carried out. In addition overexpression of eRFI protein from one of the above mutants in E. coli was performed. Purification of this protein was done by immobilized metal ion affinity chromatography (IMAC). Expression of eRF1 on the sup45 mutants through Western blot analysis suggested that the cellular levels of eRF 1 in the mutants were similar with that the wild type. The data indicated that the phenotypic of these mutants were not caused by the difference of cellular levels of eRFI but rather due to an amino acids substitution in eRFI. Overexpression of the eRFI from sup45-1 Is in the E. colt [BL21 (DE3)] through an overexpression plasmid (pITB53) was obtained. This protein was purified by the immobilized metal ion affinity chromatography. The effect of metal ions Cu e+ , Nit+ , and combination of Cu 2, with Nit+ on immobilized Chelating Sepharose Fast Flow matrix, in purification of eRFl-IIis6 did not exhibit a significant difference. To confirm that amino acid substitution of eRF1 is able to effect its functional activity, the interaction study between eRFI and other components that involved in the translational termination process need to be elucidated.