Exploring the sequence–function relationship in transcriptional regulation by the lac O1 operator

Understanding how binding of a transcription factor to an operator is influenced by the operator sequence is an ongoing quest. It facilitates discovery of alternative binding sites as well as tuning of transcriptional regulation. We investigated the behavior of the Escherichia coli Lac repressor (LacI) protein with a large set of lac O1 operator variants. The 114 variants examined contained a mean of 2.9 (range 0–4) mutations at positions −4, −2, +2 and +4 in the minimally required 17 bp operator. The relative affinity of LacI for the operators was examined by quantifying expression of a GFP reporter gene and Rosetta structural modeling. The combinations of mutations in the operator sequence created a wide range of regulatory behaviors. We observed variations in the GFP fluorescent signal among the operator variants of more than an order of magnitude under both uninduced and induced conditions. We found that a single nucleotide change may result in changes of up to six‐ and 12‐fold in uninduced and induced GFP signals, respectively. Among the four positions mutated, we found that nucleotide G at position −4 is strongly correlated with strong repression. By Rosetta modeling, we found a significant correlation between the calculated binding energy and the experimentally observed transcriptional repression strength for many operators. However, exceptions were also observed, underscoring the necessity for further improvement in biophysical models of protein–DNA interactions. To gain a predictive understanding of the influence of operator sequence variation to transcription factor binding behavior, we quantitatively investigated 114 lac O1 operator sequence variants in vivo for their ability to interact with the LacI transcription repressor. Furthermore, we attempted to rationalize the experimentally observed operator behaviors by Rosetta modeling for a subset of operator variants exhibiting distinct traits.