Using deep mutagenesis to understand genetic and physical interactions
The first aim of this thesis was to tackle a core question in biology—to understand how large numbers of mutations combine together to influence phenotypes. In order to do so, we built a combinatorially-complete library of naturally occurring variants in a yeast tRNA. For the first time in any gene,...
| Autor: | |
|---|---|
| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2020 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/668731 |
| Acceso en línea: | http://hdl.handle.net/10803/668731 |
| Access Level: | acceso abierto |
| Palabra clave: | Genetic interactions tRNA Protein-protein interactions Mutagenesis Genetics Interaccions genètiques RNAt Interaccions de proteïnes Mutagènesi Genètica 575 |
| Sumario: | The first aim of this thesis was to tackle a core question in biology—to understand how large numbers of mutations combine together to influence phenotypes. In order to do so, we built a combinatorially-complete library of naturally occurring variants in a yeast tRNA. For the first time in any gene, we could quantify the extent of which both the effects of individual mutations and the interactions between pairs of mutations change across a large number of closely-related genotypes. We found that all mutations switch from beneficial to detrimental effects and all interactions switch from positive to negative in different backgrounds. Secondly, with the use of systematic mutagenesis, protein complementation assays and deep sequencing, we developed a new experimental methodology to map the interaction interfaces of physically interacting proteins at amino acid resolution. The approach works by quantifying the effects of mutations on both protein binding and stability, resulting in a high resolution map of an interaction interface. |
|---|