Correlating in Vitro Measurements of Protein-DNA Binding Affinities with in Vivo Repression and Impact on the Growth Rate of the Host Organism

Abstract

In order to fully decode the information of patient genomes, we must determine which protein polymorphisms alter function. One resource for addressing this question is the sequence set of naturally occurring homologs. Sequence comparisons easily identify conserved positions, which usually cannot be changed without consequence. However, the impact of changing nonconserved positions is less-easily predicted. We have devised a model system for testing computational identifications of important nonconserved positions and measuring the functional impact from amino acid substitutions at these positions. The model system is based on chimeric proteins of the LacI/GalR family; functional change can be detected as either altered in vivo repression or altered in vitro DNA binding properties. For this system to accurately model a naturally-evolving system, an observed change in repression must be large enough to impact the growth of the host organism. Here, we use a series of point mutations in an engineered LacI/GalR transcription repressor to correlate altered in vivo repression with thermodynamic measurements of DNA binding affinities and bacterial growth rates. Results will determine whether most changes in repression are due to changes in DNA-binding affinity for the in vivo operator DNA binding site, and will determine how large a change in repression is required to alter bacterial life cycles.