Abstract
Mapping transcription factor (TF) binding sites along a DNA backbone is crucial in understanding the regulatory circuits that control cellular processes. Here, we deployed a method adopting bioconjugation, nanofluidic confinement and fluorescence single molecule imaging for direct mapping of TF (RNA polymerase) binding sites on field-stretched single DNA molecules. Using this method, we have mapped out five of the TF binding sites of E. coli RNA polymerase to bacteriophage λ-DNA, where two promoter sites and three pseudo-promoter sites are identified with the corresponding binding frequency of 45% and 30%, respectively. Our method is quick, robust and capable of resolving protein-binding locations with high accuracy (∼ 300 bp), making our system a complementary platform to the methods currently practiced. It is advantageous in parallel analysis and less prone to false positive results over other single molecule mapping techniques such as optical tweezers, atomic force microscopy and molecular combing, and could potentially be extended to general mapping of protein–DNA interaction sites.
Highlights
Transcription factors (TFs) are proteins that bind to specific bases of DNA using DNA-binding domains to carry out the process of transcription [1], which play a major role in the process of transcribing sequential information from DNA to messenger RNA
When more than one protein is involved in complex formation, ChIP results may not tell if the TF of interest is directly bound to the DNA sequence or through other proteins as a complex
Images were collected and analyzed and a histogram is plotted with results from ∼ 200 fluosphere–DNA–RNA polymerase (RNAP) complexes (Figure 5)
Summary
Transcription factors (TFs) are proteins that bind to specific bases of DNA using DNA-binding domains to carry out the process of transcription [1], which play a major role in the process of transcribing sequential information from DNA to messenger RNA. When more than one protein is involved in complex formation, ChIP results may not tell if the TF of interest is directly bound to the DNA sequence or through other proteins as a complex. Methods such as electrophoretic mobility shift assay (EMSA) and DNA footprinting are used for identification of TF binding sites mostly in vitro [6]. Each technique mentioned above has its own advantages and limitations and in most cases, more than one technique is employed to understand DNA– protein interactions [7,8]
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