Molecular Evolution of Posttranslational Regulation in Intrinsically Disordered Regions

Protein posttranslational regulation is a major facet of protein function, and efforts have been made to systematically characterize the level of control of proteins. For example, systematic determination of protein localization, phosphorylation, and interactions have allowed the examination of the regulatory network of the cell. However, the study of the evolution of this underlying regulatory network requires a higher resolution analysis of the sequences that are responsible for this level of control. The overarching goal of this thesis was to examine the role of the evolution of protein regulatory sequences as a molecular mechanism driving functional diversity. I developed computational tools and methods for the identification and characterization of these regulatory sequences, as well as experimental approaches to study the evolutionary impact of changes within these sequences.I first characterized the evolution of phosphorylation sites and used the property that they are strongly conserved relative to their flanking disordered regions as a computational means to systematically identify motifs in the budding yeast proteome. These results suggest that incorporating evolutionary conservation is sufficient for the prediction of around 30% of the known short linear motifs. Applying these computational approaches to the budding yeast proteome showed that thousands of short linear motifs exist and still remain uncharacterized.Using a relative rates test, I showed that motifs frequently change selective constraints after gene duplication and showed that these changes can alter protein regulation over evolution. Finally, I designed a high-throughput experimental pipeline to systematically, quantitatively and precisely assess the fitness consequences of rewiring a regulatory network and applied it to test whether a bi-functional protein has sub-functionalized over evolution.Ph.D