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The bacterial transcription factor LacI loops DNA by binding to two separatelocations on the DNA simultaneously. Despite being one of the best-studiedmodel systems for transcriptional regulation, the number and conformations ofloop structures accessible to LacI remain unclear, though the importance ofmultiple co-existing loops has been implicated in interactions between LacI andother cellular regulators of gene expression. To probe this issue, we havedeveloped a new analysis method for tethered particle motion, a versatile andcommonly-used in vitro single-molecule technique. Our method, vbTPM, performsvariational Bayesian inference in hidden Markov models. It learns the number ofdistinct states (i.e., DNA-protein conformations) directly from tetheredparticle motion data with better resolution than existing methods, while easilycorrecting 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 resultsconfirm that changes in LacI conformation and DNA binding topology bothcontribute to the repertoire of LacI-mediated loops formed in vitro, andprovide qualitatively new input for models of looping and transcriptionalregulation. We expect vbTPM to be broadly useful for probing complexprotein-nucleic acid interactions.

Multiple Lac-mediated loops revealed by Bayesian statistics and tethered particle motion