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If a TF is bound at a certain location and the binding is not functional, the probability for conservation of the binding sites across several species is low.
This finding is consistent with previous observations that TF affinity is not sufficient for TF binding, and that TF binding is not sufficient for function; in fact, previous work has shown that approximately 42% of TF binding is not functional (Gao et al., 2004).
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If a binding site is not functional, this results in reduced fitness.
While the construct lacking the miR-96*-binding site was still significantly responsive to miR-96 repression (P< 0.01), the mutant deprived of miR-96-binding site was not (Fig. 3A), indicating that MYRIP 3′UTR is regulated uniquely by miR-96, and that the predicted miR-96* binding site is not functional.
However, the binding site of R3-36/HPSE (684 to 684) is not functional; the decrease of luciferase activity is not statistically significant.
A Paris look is not functional!
If simultaneous multiple binding is not sterically possible, then apparent gains in functional affinity are likely to be small and due only to the effect of increased rebinding, which is dependent on diffusion rates and surface antigen concentration [33].
Furthermore, although computational methods can successful identify binding sites that are bound by a particular TF in vitro, most of the predicted binding sites are not functional in vivo (Li et al. 2011; Whittle et al. 2009).
However, it is also possible that some of the S. cerevisiae binding sites are not functional despite being bound in S. cerevisiae.
This could suggest that a majority of the E2F4 peaks in K562 are not functional binding sites, but we cannot exclude that our data are missing several true binding sites in HeLa-S3.
For example, most Fox binding motifs (UGCAUG) predicted in introns are not functional, particularly those that are not conserved through evolution ([53] and Benoit Chabot, personal communication).
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