title: Multiple LacI-mediated loops revealed by Bayesian statistics and tethered particle motion.
abstract: The bacterial transcription factor LacI loops DNA by binding to two separate locations on the DNA simultaneously. Despite being one of the best-studied model systems for transcriptional regulation, the number and conformations of loop structures accessible to LacI remain unclear, though the importance of multiple coexisting loops has been implicated in interactions between LacI and other cellular regulators of gene expression. To probe this issue, we have developed a new analysis method for tethered particle motion, a versatile and commonly used in vitro single-molecule technique. Our method, vbTPM, performs variational Bayesian inference in hidden Markov models. It learns the number of distinct states (i.e. DNA-protein conformations) directly from tethered particle motion data with better resolution than existing methods, while easily correcting for common experimental artifacts. Studying short (roughly 100 bp) LacI-mediated loops, we provide evidence for three distinct loop structures, more than previously reported in single-molecule studies. Moreover, our results confirm that changes in LacI conformation and DNA-binding topology both contribute to the repertoire of LacI-mediated loops formed in vitro, and provide qualitatively new input for models of looping and transcriptional regulation. We expect vbTPM to be broadly useful for probing complex protein-nucleic acid interactions.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, California Institute of Technology, 1200 E. California Blvd., Pasadena, California 91125.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Johnson
authors.affiliation: Department of Statistics, Columbia University, 1255 Amsterdam Avenue MC 4690, New York, New York 10027.
authors.forename: Jan-Willem
authors.initials: JW
authors.lastname: van de Meent
authors.affiliation: Departments of Applied Physics and Biology, California Institute of Technology, 1200 E. California Blvd., Pasadena, California 91125.
authors.forename: Rob
authors.initials: R
authors.lastname: Phillips
authors.affiliation: Department of Applied Physics and Applied Mathematics, Columbia University, 200 S.W. Mudd, 500 W. 120th St. MC 4701, New York, New York 10027.
authors.forename: Chris H
authors.initials: CH
authors.lastname: Wiggins
authors.affiliation: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius väg 16C, SE-106 91 Stockholm, Sweden Department of Cell and Molecular Biology, Uppsala University, Box 256, SE-751 05 Uppsala, Sweden martin.linden@icm.uu.se.
authors.forename: Martin
authors.identifier: http://orcid.org/0000-0003-4200-0191
authors.initials: M
authors.lastname: Lindén
mesh_terms: D016477:Artifacts; D001499:Bayes Theorem; D004247:DNA / Q000737:chemistry*; D007700:Kinetics; D056968:Lac Repressors / Q000737:chemistry / Q000378:metabolism*; D008390:Markov Chains; D009038:Motion; D009690:Nucleic Acid Conformation
chemical_list: D056968:Lac Repressors; D004247:DNA