The functional role of ASP50 in protein synthesis elongation factor Tu
| dc.contributor | Graduate Program in Molecular Biology and Genetics. | |
| dc.contributor.advisor | Bilgin, Neşe. | |
| dc.contributor.author | Baş, Levent. | |
| dc.date.accessioned | 2023-03-16T11:26:16Z | |
| dc.date.available | 2023-03-16T11:26:16Z | |
| dc.date.issued | 2013. | |
| dc.description.abstract | Elongation factor Tu is an essential enzyme responsible for delivering aminoacyltRNA's to ribosomal A-site during protein synthesis. In its active GTP-bound state, it forms an EF-Tu · GTP · aminoacyl-tRNA ternary complex whereas it is inactive in the GDP-bound state. One very unstable and dynamic structural element in the nucleotide binding domain of EF-Tu is the Switch I region, whose structure changes from a β-sheet to an α-helix during the transition from the GTP- to GDP-bound state of EF-Tu. In this thesis, the role of the universally conserved Asp50 residue in the Switch I region in Escherichia coli EF-Tu was studied. Mutants of Asp50 to alanine and asparagine were made in EF-Tu and mutant EF-Tu’s were studied in their interactions with GDP, GTP and aminoacyl- tRNA, as well as in their activity in poly(Phe) synthesis, GTP hydrolysis and dipeptide formation using in vitro kinetic assays. D50A and D50N mutants of EF-Tu were comparable to wild-type EF-Tu with respect to GDP binding and GDP/GTP nucleotide exchange rates. However, the Asp50 mutants have significantly reduced affinity for aminoacyl- tRNA. When analyzed in the presence of high excess of aminoacyl-tRNA, Asp50 mutants were comparable to wild-type EF-Tu in their activity in poly(Phe) synthesis. On the other hand, pre-steady state measurements of GTP hydrolysis and peptide bond formation indicated that the amount of GTP hydrolyzed by the mutants are significantly reduced compared to wild type EF-Tu, even though the rate of GTP hydrolysis was not altered. The results obtained from this work suggest that Asp50 is crucial for a tight GTP conformation of EF-Tu. When Asp50 is mutated, the enzyme easily switches to GDP conformation, explaining the reduced affinity of the Asp50 mutants in aminoacyl-tRNA binding. On the other hand, when ternary complexes with Asp50 mutants reach to the A-site on the ribosomes, some mutant EF-Tu's may switch to their GDP-like conformation, therefore, releasing the aminoacyl-tRNA, which proceed to peptide bond formation without GTP hydrolysis. | |
| dc.format.extent | 30 cm. | |
| dc.format.pages | xv, 59 leaves ; | |
| dc.identifier.other | BIO 2013 B37 | |
| dc.identifier.uri | https://digitalarchive.library.bogazici.edu.tr/handle/123456789/15470 | |
| dc.publisher | Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2013. | |
| dc.subject.lcsh | Proteins -- Synthesis. | |
| dc.subject.lcsh | Genetic transcription. | |
| dc.title | The functional role of ASP50 in protein synthesis elongation factor Tu |
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