Until now, it has been reasonably assumed that specific base-pair recognition is the only mechanism controlling the specificity of transcription factor (TF)−DNA binding. Contrary to this assumption, here we show that nonspecific DNA sequences possessing certain repeat symmetries, when present outside of specific TF binding sites (TFBSs), statistically control TF−DNA binding preferences. We used highthroughput protein−DNA binding assays to measure the binding levels and free energies of binding for several human TFs to tens of thousands of short DNA sequences with varying repeat symmetries. Based on statistical mechanics modeling, we identify a new protein−DNA binding mechanism induced by DNA sequence symmetry in the absence of specific base-pair recognition, and experimentally demonstrate that this mechanism indeed governs protein−DNA binding preferences. protein−DNA binding is an important biophysical mechanism operating in a living cell (1). This seminal work makes it possible to interpret experiments that measured how transcription factors (TFs) search for their specific target sites flanked by nonconsensus sequence elements (1-10). A specific consensus motif is a short DNA sequence, typically 6-20 base pairs (bp), that possesses an enhanced binding affinity for a particular TF. For example, the sequence CACGTG represents the specific consensus motif for the human protein Max used in this study (Fig. 1). The process of establishing specific, consensus protein−DNA binding requires the formation of precise geometrical fit between the protein and its consensus DNA motif, accompanied by the formation of specific hydrogen and electrostatic contacts at the protein−DNA binding interface (6, 7) ( Fig. 1). In addition to binding to their consensus DNA motifs, transcription factors can also bind, albeit with lower affinity, to DNA regions lacking any consensus motifs. The term "nonspecific protein−DNA binding" (6) is typically used to describe these weaker interactions. Von Hippel and Berg suggested classifying nonspecific protein−DNA binding into two related mechanisms (6). The first mechanism includes protein binding to its mutated specific motifs that retain some residual, reduced specificity. The second mechanism is largely DNA sequence independent, and it involves electrostatic binding modulated by the overall DNA geometry (6). Despite significant experimental progress, molecular mechanisms responsible for these two types of nonspecific binding remain poorly understood, and the free energy of nonspecific protein−DNA binding has not been systematically characterized (11)(12)(13)(14). The interplay between consensus and nonconsensus DNA sequence elements emerges as a dominant factor that governs protein−DNA binding preferences. However, this interplay is also poorly understood (15, 16). Until now, it has been reasonably assumed that specific (consensus) base-pair recognition must control the genome-wide specificity of TF−DNA binding.Contrary to this assumption, here we identify a general mechanism for protein−DNA bi...

A mutation in the allosteric site of the caspase 3 dimer interface of Val266 to histidine abolishes activity of the enzyme, and models predict that the mutation mimics the action of small molecule allosteric inhibitors by preventing formation of the active site. Mutations were coupled to His266 at two sites in the interface, E124A and Y197C. We present results from X-ray crystallography, enzymatic activity and molecular dynamics simulations for seven proteins, consisting of single, double and triple mutants. The results demonstrate that considering allosteric inhibition of caspase 3 as a shift between discrete ‘off-state’ or ‘on-state’ conformations is insufficient. Although His266 is accommodated in the interface, the structural defects are propagated to the active site through a helix on the protein surface. A more comprehensive view of allosteric regulation of caspase 3 requires the representation of an ensemble of inactive states and shows that subtle structural changes lead to the population of the inactive ensemble.

SUMMARY Paralogous transcription factors (TFs) are oftentimes reported to have identical DNA-binding motifs, despite the fact that they perform distinct regulatory functions. Differential genomic targeting by paralogous TFs is generally assumed to be due to interactions with protein co-factors or the chromatin environment. Using a computational-experimental framework called iMADS (integrative modeling and analysis of differential specificity), we show that, contrary to previous assumptions, paralogous TFs bind differently to genomic target sites even in vitro. We used iMADS to quantify, model, and analyze specificity differences between 11 TFs from 4 protein families. We found that paralogous TFs have diverged mainly at mediumand low-affinity sites, which are poorly captured by current motif models. We identify sequence and shape features differentially preferred by paralogous TFs, and we show that the intrinsic differences in specificity among paralogous TFs contribute to their differential in vivo binding. Thus, our study represents a step forward in deciphering the molecular mechanisms of differential specificity in TF families.

BackgroundThe Myc-Max heterodimer is a transcription factor that regulates expression of a large number of genes. Genome occupancy of Myc-Max is thought to be driven by Enhancer box (E-box) DNA elements, CACGTG or variants, to which the heterodimer binds in vitro.ResultsBy analyzing ChIP-Seq datasets, we demonstrate that the positions occupied by Myc-Max across the human genome correlate with the RNA polymerase II, Pol II, transcription machinery significantly better than with E-boxes. Metagene analyses show that in promoter regions, Myc is uniformly positioned about 100 bp upstream of essentially all promoter proximal paused polymerases with Max about 15 bp upstream of Myc. We re-evaluate the DNA binding properties of full length Myc-Max proteins. Electrophoretic mobility shift assay results demonstrate Myc-Max heterodimers display significant sequence preference, but have high affinity for any DNA. Quantification of the relative affinities of Myc-Max for all possible 8-mers using universal protein-binding microarray assays shows that sequences surrounding core 6-mers significantly affect binding. Compared to the in vitro sequence preferences, Myc-Max genomic occupancy measured by ChIP-Seq is largely, although not completely, independent of sequence specificity.ConclusionsWe quantified the affinity of Myc-Max to all possible 8-mers and compared this with the sites of Myc binding across the human genome. Our results indicate that the genomic occupancy of Myc cannot be explained by its intrinsic DNA specificity and suggest that the transcription machinery and associated promoter accessibility play a predominant role in Myc recruitment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0482-3) contains supplementary material, which is available to authorized users.

Background: The Myc-Max heterodimer is a transcription factor that regulates expression of a large number of genes. Genome occupancy of Myc-Max is thought to be driven by Enhancer box (E-box) DNA elements, CACGTG or variants, to which the heterodimer binds in vitro. Results: By analyzing ChIP-Seq datasets, we demonstrate that the positions occupied by Myc-Max across the human genome correlate with the RNA polymerase II, Pol II, transcription machinery significantly better than with E-boxes. Metagene analyses show that in promoter regions, Myc is uniformly positioned about 100 bp upstream of essentially all promoter proximal paused polymerases with Max about 15 bp upstream of Myc. We re-evaluate the DNA binding properties of full length Myc-Max proteins. Electrophoretic mobility shift assay results demonstrate Myc-Max heterodimers display significant sequence preference, but have high affinity for any DNA. Quantification of the relative affinities of Myc-Max for all possible 8-mers using universal protein-binding microarray assays shows that sequences surrounding core 6-mers significantly affect binding. Compared to the in vitro sequence preferences, Myc-Max genomic occupancy measured by ChIP-Seq is largely, although not completely, independent of sequence specificity.