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. 2002 Mar 1;30(5):1255-61.
doi: 10.1093/nar/30.5.1255.

Nucleotides of transcription factor binding sites exert interdependent effects on the binding affinities of transcription factors

Martha L Bulyk  1 , Philip L F Johnson, George M Church
Affiliations

Nucleotides of transcription factor binding sites exert interdependent effects on the binding affinities of transcription factors

Martha L Bulyk et al. Nucleic Acids Res. .

Abstract

We can determine the effects of many possible sequence variations in transcription factor binding sites using microarray binding experiments. Analysis of wild-type and mutant Zif268 (Egr1) zinc fingers bound to microarrays containing all possible central 3 bp triplet binding sites indicates that the nucleotides of transcription factor binding sites cannot be treated independently. This indicates that the current practice of characterizing transcription factor binding sites by mutating individual positions of binding sites one base pair at a time does not provide a true picture of the sequence specificity. Similarly, current bioinformatic practices using either just a consensus sequence, or even mononucleotide frequency weight matrices to provide more complete descriptions of transcription factor binding sites, are not accurate in depicting the true binding site specificities, since these methods rely upon the assumption that the nucleotides of binding sites exert independent effects on binding affinity. Our results stress the importance of complete reference tables of all possible binding sites for comparing protein binding preferences for various DNA sequences. We also show results suggesting that microarray binding data using particular subsets of all possible binding sites can be used to extrapolate the relative binding affinities of all possible full-length binding sites, given a known binding site for use as a starting sequence for site preference refinement.

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Figures

Figure 1
Figure 1
Model depicting interactions between the Zif268 phage display library and the DNA used in microarray binding experiments. The three zinc fingers of Zif268 (F1, F2 and F3) are aligned to show contacts to the nucleotides of the DNA binding site as inferred from the crystal structure of Zif268 and biochemical experiments. The zinc finger amino acid positions are numbered relative to the first helical residue (position 1). The randomized positions in the α-helix of the second finger are circled. DNA base pairs marked N were fixed as particular sequences (11). © Copyright (2001) National Academy of Sciences of the USA.
Figure 2
Figure 2
Zero order HMM using an alphabet of two nucleotides (A,T) for clarity. Circles represent states; arrows represent transitions. The numbers alongside the arrows specify transition probabilities. The emission distribution for each state (except for the silent start state) is contained within each circle. If this were a real zero order model, the four emitting states would contain distributions over all four nucleotides. Adapted from Durbin et al. (8).
Figure 3
Figure 3
First order HMM using an alphabet of two nucleotides (A,T) for clarity. Circles represent states; arrows represent transitions. The letter inside each circle is the only nucleotide emitted by that state (at 100% probability). To represent the full first order model, there would be four states for each position. Adapted from Durbin et al. (8).
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