(169f) Engineering Transcription Factors with Novel DNA-Binding Specificity Using Comparative Genomics

Transcription factors must have exquisite DNA-binding specificity to properly control gene expression. Remarkably, genetic and structural data suggest that only a few key amino acid residues are involved in determining this specificity. By changing these residues, it is possible to change the DNA-binding specificity of these proteins. However, simple rules that explain this relationship a priori do not currently exist. Identifying these rules will be useful for engineering novel transcription factors with applications in metabolic engineering, gene therapy, and synthetic biology.

In this research, we explore the use of comparative genomics for engineering transcription factors with novel DNA-binding specificity. By identifying conserved amino acids in the DNA binding domain of orthologous transcription factors in different phyletic lineages, we can infer relations between different amino acid clusters and their cognate DNA sequence for different families of transcription factors. We have applied this approach to the CRP/FNR family of winged helix-turn-helix transcription factors. For this family of transcription factors, we were able to computationally identify eight mutations that are predicted to lead to novel DNA specificity. These computational predictions were tested experimentally using the CRP protein from Escherichia coli. Of the eight, four mutations were shown to have novel DNA-binding specificity in vivo. These results demonstrate the power of comparative genomics and sequence-based analysis for protein engineering. We are currently refining our models to improve the success rate of our predictions and applying our approach to other families of transcription factors.