Rationalization of Gene Regulation by a Eukaryotic Transcription Factor: Calculation of Regulatory Region Occupancy from Predicted Binding Affinities

DNA-binding proteins regulate gene expression by binding preferentially to a set of related sequences. In order to quantify the correlation between gene regulation and the presence of sequence motifs, the affinity of a transcription factor for each variant of the binding site must be known or predicted. In addition, the contribution of multiple binding sites to the regulation of a single gene must be modeled. To predict the affinity of the yeast Leu3 transcription factor for genomic-binding sites, we measured the in vitro equilibrium dissociation constants of 43 binding-site variants and established that the free energy of binding can be approximated as a sum of free energy contributions from each base-pair. This allows the prediction of an equilibrium dissociation constant for all potential binding sites in the genome and, therefore, their fractional occupancy at some assumed concentration of free Leu3. From the occupancy of individual sites, the probability that at least one site is occupied within a defined segment upstream of a gene was calculated for all genes in yeast. We find that this probability is substantially better correlated with regulation by Leu3 than is the number of binding sites. This is true whether the number of binding sites is based on a consensus site definition of the binding site or by enumeration of all variants that have a predicted Kd value below some threshold. The occupancy calculation was best able to rationalize the Leu3-regulated gene set over a Leu3 concentration range that spans the Kd values for the best sites.