Single-Molecule Detection of Transcription Factor Binding to DNA in Real Time:
Specificity, Equilibrium, and Kinetic Parameters
Eric A. Nalefski
Eugene Nebelitsky
Janice A. Lloyd
Steven R. Gullans
10.1021/bi0602011.s001
https://acs.figshare.com/articles/journal_contribution/Single_Molecule_Detection_of_Transcription_Factor_Binding_to_DNA_in_Real_Time_Specificity_Equilibrium_and_Kinetic_Parameters/3046927
Specificity and temporal control of transcriptional machinery are encoded within sequence-specific transcription factors, of which there are thousands in mammalian genomes. Efforts to completely
decipher this code will require an understanding of the DNA binding thermodynamic and kinetic properties
displayed by each transcription factor, a daunting task given the current methodologies for measuring
these interactions. Here, we present a novel methodology to quantify the binding of proteins to target
DNA molecules based on single-molecule detection and real-time counting of individual free and bound
fluorescently tagged molecules flowing past a detection device. Using this technology, we measured DNA
binding by fluorescently tagged domains of four distinct transcription factors, namely, human early growth
response protein Egr-1, vertebrate GATA-1, <i>Drosophila</i> GAGA factor, and λ bacteriophage Cro repressor.
These proteins represent different structural classes (zinc-finger and helix−turn−helix), quaternary states
(monomeric and dimeric), and relative affinities (high, intermediate, and low). Specific binding of each
protein to its cognate DNA target was demonstrated at low picomolar concentrations. The equilibrium
(<i>K</i><sub>d</sub>) and kinetic (<i>k</i><sub>on</sub> and <i>k</i><sub>off</sub>) constants governing DNA binding by one of these transcription factors, that
of Egr-1, were measured using this approach. <i>K</i><sub>d</sub> values obtained from three different types of saturation
titrations were reproducible and consistent, yielding values between 10 and 14 pM that, along with the
kinetic constants, agree closely with literature values. Because this methodology offers several significant
advantages over other existing approaches, namely, real-time determination, requirement for small amounts
of reagents, high reproducibility, exquisite sensitivity, and amenability to high-throughput analysis, it is
suitable for characterizing DNA-binding proteins as well as other interacting pairs of molecules that can
be fluorescently tagged.
2006-11-21 00:00:00
fluorescently
λ bacteriophage Cro repressor
GATA
transcription factors
target DNA molecules
DNA binding
Transcription Factor Binding
protein
methodology
GAGA