Abstract
BackgroundThe spatial organization of transcription factor binding sites in regulatory DNA, and the composition of intersite sequences, influences the assembly of the multiprotein complexes that regulate RNA polymerase recruitment and thereby affects transcription. We have developed a genetic approach to investigate how reporter gene transcription is affected by varying the spacing between transcription factor binding sites. We characterized the components of promoter architecture that govern the yeast transcription factors Cbf1 and Met31/32, which bind independently, but collaboratively recruit the coactivator Met4.ResultsA Cbf1 binding site was required upstream of a Met31/32 binding site for full reporter gene expression. Distance constraints on coactivator recruitment were more flexible than those for cooperatively binding transcription factors. Distances from 18 to 50 bp between binding sites support efficient recruitment of Met4, with only slight modulation by helical phasing. Intriguingly, we found that certain sequences located between the binding sites abolished gene expression.ConclusionThese results yield insight to the influence of both binding site architecture and local DNA flexibility on gene expression, and can be used to refine computational predictions of gene expression from promoter sequences. In addition, our approach can be applied to survey promoter architecture requirements for arbitrary combinations of transcription factor binding sites.
Highlights
The spatial organization of transcription factor binding sites in regulatory DNA, and the composition of intersite sequences, influences the assembly of the multiprotein complexes that regulate RNA polymerase recruitment and thereby affects transcription
Our goal is to understand how promoter architectures differ for transcription factors that participate in coactivator recruitment, versus those that bind cooperatively
We discovered that distance constraints on coactivator recruitment were more flexible than those for cooperatively binding transcription factors
Summary
The spatial organization of transcription factor binding sites in regulatory DNA, and the composition of intersite sequences, influences the assembly of the multiprotein complexes that regulate RNA polymerase recruitment and thereby affects transcription. We characterized the components of promoter architecture that govern the yeast transcription factors Cbf and Met31/32, which bind independently, but collaboratively recruit the coactivator Met. When bound to DNA, transcription factors anchor the assembly of multiprotein complexes that influence the recruitment of RNA polymerase. Since transcription factors recognize specific sites on DNA, the distance between these binding sites can influence how transcription factors interact with each other and other proteins. Overlapping sites may prevent two transcription factors from binding simultaneously, while sites too distant from each other may hinder bound transcription factors from recruiting (page number not for citation purposes). A binding site was considered conserved if invariant copies were aligned in at least 3 out of 4 closely-related Saccharomyces species in a multiple sequence alignment [24, 49]
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