A quantitative understanding of lac repressor’s binding specificity and flexibility

Abstract

Lac repressor, the first discovered transcriptional regulator, has been shown to confer multiple modes of binding to its operator sites depending on the central spacer length. Other homolog members in the LacI/GalR family (PurR and YcjW) cannot bind their operator sites with similar structural flexibility. To decipher the underlying mechanism for this unique property, we used Spec-seq approach combined with site-directed mutagenesis to quantify the DNA binding specificity of multiple hybrids of lacI and PurR. We find that lac repressor’s recognition di-residues YQ and its hinge helix loop regions are both critical for its structural flexibility. Also, specificity profiling of the whole lac operator suggests that a simple additive model from single variants suffice to predict other multivariant sites’ energy reasonably well, and the genome occupancy model based on this specificity data correlates well with in vivo lac repressor binding profile.

Graphical abstract

Keywords

lac repressor / binding flexibility / Spec-seq / ionic strength

References

[1] Jacob, F. and Monod, J. (1961) Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol., 3, 318–356

[2] Lewis, M. (2005) The lac repressor. C. R. Biol., 328, 521–548

[3] Gilbert, W. and Maxam, A. (1973) The nucleotide sequence of the lac operator. Proc. Natl. Acad. Sci. USA, 70, 3581–3584

[4] Zuo, Z. and Stormo, G. D. (2014) High-resolution specificity from DNA sequencing highlights alternative modes of lac repressor binding. Genetics, 198, 1329–1343

[5] Mossing, M. C. and Record, M. T. Jr. (1985) Thermodynamic origins of specificity in the lac repressor-operator interaction: Adaptability in the recognition of mutant operator sites. J. Mol. Biol., 186, 295–305

[6] Frank, D. E., Saecker, R. M., Bond, J. P., Capp, M. W., Tsodikov, O. V., Melcher, S. E., Levandoski, M. M. and Record, M. T. Jr. (1997) Thermodynamics of the interactions of lac repressor with variants of the symmetric lac operator: effects of converting a consensus site to a non-specific site. J. Mol. Biol., 267, 1186–1206

[7] Hart, D. J., Speight, R. E., Cooper, M. A., Sutherland, J. D. and Blackburn, J. M. (1999) The salt dependence of DNA recognition by NF-κB p50: a detailed kinetic analysis of the effects on affinity and specificity. Nucleic Acids Res., 27, 1063–1069

[8] Benos, P. V., Bulyk, M. L. and Stormo, G. D. (2002) Additivity in protein-DNA interactions: how good an approximation is it? Nucleic Acids Res., 30, 4442–4451

[9] Stormo, G. D. (2013) Modeling the specificity of protein-DNA interactions. Quant. Biol., 1, 115–130

[10] Maerkl, S. J. and Quake, S. R. (2007) A systems approach to measuring the binding energy landscapes of transcription factors. Science, 315, 233–237

[11] Novichkov, P. S., Laikova, O. N., Novichkova, E. S., Gelfand, M. S., Arkin, A. P., Dubchak, I. and Rodionov, D. A. (2010) RegPrecise: a database of curated genomic inferences of transcriptional regulatory interactions in prokaryotes. Nucleic Acids Res., 38, D111–D118

[12] Daber, R. and Lewis, M. (2009) Towards evolving a better repressor. Protein Eng. Des. Sel., 22, 673–683

[13] Record, M. T. Jr, deHaseth, P. L. and Lohman, T. M. (1977) Interpretation of monovalent and divalent cation effects on the lac repressor-operator interaction. Biochemistry, 16, 4791–4796

[14] von Hippel, P. H. (2014) Increased subtlety of transcription factor binding increases complexity of genome regulation. Proc. Natl. Acad. Sci. USA, 111, 17344–17345

[15] Schumacher, M. A., Choi, K. Y., Zalkin, H. and Brennan, R. G. (1994) Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices. Science, 266, 763–770

[16] Lewis, M., Chang, G., Horton, N. C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G. and Lu, P. (1996) Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science, 271, 1247–1254

[17] Romanuka, J., Folkers, G. E., Biris, N., Tishchenko, E., Wienk, H., Bonvin, A. M., Kaptein, R. and Boelens, R. (2009) Specificity and affinity of Lac repressor for the auxiliary operators O2 and O3 are explained by the structures of their protein-DNA complexes. J. Mol. Biol., 390, 478–489

[18] Milk, L., Daber, R. and Lewis, M. (2010) Functional rules for lac repressor-operator associations and implications for protein-DNA interactions. Protein Sci., 19, 1162–1172

[19] Kalodimos, C. G., Boelens, R. and Kaptein, R. (2004) Toward an integrated model of protein-DNA recognition as inferred from NMR studies on the Lac repressor system. Chem. Rev., 104, 3567–3586

[20] Riggs, A. D., Suzuki, H. and Bourgeois, S. (1970) Lac repressor-operator interaction: I. Equilibrium studies. J. Mol. Biol., 48, 67–83

[21] von Hippel, P. H. (2004) Completing the view of transcriptional regulation. Science, 305, 350–352

[22] Cournac, A. and Plumbridge, J. (2013) DNA looping in prokaryotes: experimental and theoretical approaches. J. Bacteriol., 195, 1109–1119

[23] Gama-Castro, S., Salgado, H., Peralta-Gil, M., Santos-Zavaleta, A., Muñiz-Rascado, L., Solano-Lira, H., Jimenez-Jacinto, V., Weiss, V., García-Sotelo, J. S., López-Fuentes, A., (2011) RegulonDB version 7.0: transcriptional regulation of Escherichia coli K-12 integrated within genetic sensory response units (Gensor Units). Nucleic Acids Res., 39, D98–D105

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