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

Abstract

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 O(1) 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.

DOI: 10.1111/j.1742-4658.2012.08635.x

Cite this paper

@article{Maity2012ExploringTS, title={Exploring the sequence-function relationship in transcriptional regulation by the lac O1 operator.}, author={Tuhin Subhra Maity and Ramesh Kumar Jha and Charlie E. M. Strauss and John Dunbar}, journal={The FEBS journal}, year={2012}, volume={279 14}, pages={2534-43} }