Molecular Mechanisms for the Evolution of DNA Specificity in a Transcription Factor Family
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Transcription factors (TFs) bind to specific DNA sequences near target genes to precisely coordinate their regulation. Despite the central role of transcription factors in development and homeostasis, the mechanisms by which TFs have evolved to bind and regulate distinct DNA sequences are poorly understood. This dissertation details the highly collaborative work to determine the genetic, biochemical and biophysical mechanisms by which distinct DNA-binding specificities evolved in the steroid receptor (SR) family of transcription factors. Using ancestral protein reconstruction, we resurrected and functionally characterized the historical transition in DNA-binding specificity between ancient SR proteins. We found that DNA-binding specificity evolved by changes in the energetic components of binding; interactions at the protein-DNA interface were weakened while inter-protein cooperativity was greatly improved. We identified a group of fourteen historical substitutions that were sufficient to recapitulate the derived protein's binding function. Three of these substitutions, which we defined as function-switching, were sufficient to change DNA specificity; however, their introduction greatly decreased binding affinity and was deleterious for protein function. A group of eleven permissive substitutions, which had no effect on DNA specificity, allowed for the protein to tolerate the deleterious effects of the function-switching substitutions. They non-specifically increased binding affinity by improving interactions at the protein-DNA interface and increasing inter-protein cooperativity. We then dissected the functional role of individual substitutions in both the function-switching and permissive groups. We first determined the binding affinity of all possible combinations of function-switching substitutions for a library of DNA sequences. This allowed for us to functionally characterize the sequence space that separated the ancestral and derived DNA-binding specificities as well as identify the genetic determinants for DNA specificity. Lastly, we dissected the effects of the permissive substitutions on the energetics of DNA binding to determine the mechanisms by which they exerted their permissive effect. Together, this work provides insight into the molecular determinants of DNA specificity and identifies the molecular mechanisms by which these interactions changed during the evolution of novel specificity in an important transcription factor family. This dissertation includes previously published and unpublished co-authored material.