Siggers, TrevorDuyzend, Michael HReddy, JessicaKhan, SidraBulyk, Martha2014-03-012011Siggers, Trevor, Michael H Duyzend, Jessica Reddy, Sidra Khan, and Martha L Bulyk. 2011. “Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex.” Molecular Systems Biology 7 (1): 555. doi:10.1038/msb.2011.89. http://dx.doi.org/10.1038/msb.2011.89.1744-4292http://nrs.harvard.edu/urn-3:HUL.InstRepos:11855816Recruitment of cofactors to specific DNA sites is integral for specificity in gene regulation. As a model system, we examined how targeting and transcriptional control of the sulfur metabolism genes in Saccharomyces cerevisiae is governed by recruitment of the transcriptional co-activator Met4. We developed genome-scale approaches to measure transcription factor (TF) DNA-binding affinities and cofactor recruitment to >1300 genomic binding site sequences. We report that genes responding to the TF Cbf1 and cofactor Met28 contain a novel ‘recruitment motif' (RYAAT), adjacent to Cbf1 binding sites, which enhances the binding of a Met4–Met28–Cbf1 regulatory complex, and that abrogation of this motif significantly reduces gene induction under low-sulfur conditions. Furthermore, we show that correct recognition of this composite motif requires both non-DNA-binding cofactors Met4 and Met28. Finally, we demonstrate that the presence of an RYAAT motif next to a Cbf1 site, rather than Cbf1 binding affinity, specifies Cbf1-dependent sulfur metabolism genes. Our results highlight the need to examine TF/cofactor complexes, as novel specificity can result from cofactors that lack intrinsic DNA-binding specificity.en-UScofactorDNA-binding affinitiesDNA binding sitesulfur metabolismtranscription factorJournal Article2014-03-0110.1038/msb.2011.89