Abstract
PU.1 is an essential transcription factor in normal hematopoietic lineage development. It recognizes a large number of promoter sites differing only in bases flanking a core consensus of 5'-GGAA-3'. DNA binding is mediated by its ETS domain, whose sequence selectivity directly corresponds to the transactivational activity and frequency of binding sites for full-length PU.1 in vivo. To better understand the basis of sequence discrimination, we characterized its binding properties to a high affinity and low affinity site. Despite sharing a homologous structural framework as confirmed by DNase I and hydroxyl radical footprinting, the two complexes exhibit striking heterogeneity in terms of hydration properties. High affinity binding is destabilized by osmotic stress, whereas low affinity binding is insensitive. Dimethyl sulfate footprinting showed that the major groove at the core consensus is protected in the high affinity complex but accessible in the low affinity one. Finally, destabilization of low affinity binding by salt is in quantitative agreement with the number of phosphate contacts but is substantially attenuated in high affinity binding. These observations support a mechanism of sequence discrimination wherein specifically bound water molecules couple flanking backbone contacts with base-specific interactions in a sequestered cavity at the core consensus. The implications of this model with respect to other ETS paralogs are discussed.
Highlights
PU.1 recognizes a large number of binding sites with differing affinities
We have carried out a detailed comparison of the structural and binding properties of a high affinity, low affinity, and nonspecific PU.1
ETS/DNA Interface to Osmolytes—Based on our interpretation of the osmotic stress data, the low affinity [ϩ]TG complex, which is insensitive to osmolyte, should lack the asymmetrically accessible compartment found with the [Ϫ]GC site
Summary
PU. recognizes a large number of binding sites with differing affinities. Results: The role of hydration in sequence-specific binding by the ETS domain of PU. was determined. Destabilization of low affinity binding by salt is in quantitative agreement with the number of phosphate contacts but is substantially attenuated in high affinity binding These observations support a mechanism of sequence discrimination wherein bound water molecules couple flanking backbone contacts with base-specific interactions in a sequestered cavity at the core consensus. PU. (or Spi-1) is a lineage-restricted member of the ETS family of transcription factors that shares a structurally conserved DNA-binding domain It binds a large number of cognate sequences (mostly of myeloid and lymphoid origin) harboring a central 5Ј-GGAA-3Ј consensus [1, 2]. In the co-crystal structure of the ETS domain of PU. bound to a high affinity binding site [8], the recognition helix makes base-specific contacts (several of which are water-mediated) in the major groove of the 5Ј-GGAA-3Ј core consensus. We propose a model of sequence-specific discrimination wherein flanking backbone contacts are coupled to basespecific interactions between the recognition helix and the core consensus via a network of bound water molecules
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