Abstract
Physicochemical properties of DNA, such as shape, affect protein-DNA recognition. However, the properties of DNA that are most relevant for predicting the binding sites of particular transcription factors (TFs) or classes of TFs have yet to be fully understood. Here, using a model that accurately captures the melting behavior and breathing dynamics (spontaneous local openings of the double helix) of double-stranded DNA, we simulated the dynamics of known binding sites of the TF and nucleoid-associated protein Fis in Escherichia coli. Our study involves simulations of breathing dynamics, analysis of large published in vitro and genomic datasets, and targeted experimental tests of our predictions. Our simulation results and available in vitro binding data indicate a strong correlation between DNA breathing dynamics and Fis binding. Indeed, we can define an average DNA breathing profile that is characteristic of Fis binding sites. This profile is significantly enriched among the identified in vivo E. coli Fis binding sites. To test our understanding of how Fis binding is influenced by DNA breathing dynamics, we designed base-pair substitutions, mismatch, and methylation modifications of DNA regions that are known to interact (or not interact) with Fis. The goal in each case was to make the local DNA breathing dynamics either closer to or farther from the breathing profile characteristic of a strong Fis binding site. For the modified DNA segments, we found that Fis-DNA binding, as assessed by gel-shift assay, changed in accordance with our expectations. We conclude that Fis binding is associated with DNA breathing dynamics, which in turn may be regulated by various nucleotide modifications.
Highlights
Transcription factors (TFs), which play an important role in myriad cellular processes, are proteins that regulate gene expression through specific interactions with DNA at cis regulatory sites [1,2,3]
We report that the propensity of transient opening and re-closing of the double helix, resulting from thermal fluctuations, aka ‘‘DNA breathing’’ or ‘‘DNA bubbles,’’ can be associated with binding affinity in the case of Fis, a well-studied nucleoid-associated protein in Escherichia coli
We found that a particular breathing profile is characteristic of highaffinity Fis binding sites and that DNA fragments known to bind Fis in vivo are statistically enriched for this profile
Summary
Transcription factors (TFs), which play an important role in myriad cellular processes, are proteins that regulate gene expression through specific interactions with DNA at cis regulatory sites [1,2,3]. The binding sites of a TF can be mapped on a genome-wide scale using various methods, such as chromatin immunoprecipitation followed by either microarray analysis (ChIP-chip) or sequencing (ChIP-seq) [5]. Another approach is protein binding microarray (PBM) analysis [6]. Application of these methods has revealed that the binding sites of a TF can be numerous, diverse, and difficult to represent with conventional models for TF binding sites [2,7,8], such as a consensus sequence or position weight matrix (PWM) model [9]. A possible explanation for these findings is that models neglect physicochemical features of DNA that are important for protein-DNA recognition
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