The Biophysical Basis for Phosphorylation-Enhanced DNA-Binding Autoinhibition of the ETS1 Transcription Factor.

The eukaryotic transcription factor ETS1 is regulated by an intrinsically disordered serine-rich region (SRR) that transiently associates with the adjacent ETS domain to inhibit DNA binding. In this study, we further elucidated the physicochemical basis for ETS1 autoinhibition by characterizing the interaction of its ETS domain with a series of synthetic peptides corresponding to the SRR. Binding is driven by the hydrophobic effect and enhanced electrostatically by phosphorylation of serines adjacent to aromatic residues in the amphipathic SRR. Structural characterization of the dynamic peptide/protein complex by NMR spectroscopy and X-ray crystallography revealed multiple modes of binding that lead to autoinhibition by synergistically blocking the DNA-binding interface of the ETS domain and stabilizing an appended helical inhibitory module against allosterically induced unfolding. Consistent with these conclusions, the SRR peptide does not interact with DNA-bound ETS1. In addition, we found that the ETS1 SRR phosphopeptide binds to distantly related PU.1 in vitro, indicating that autoinhibition exploits features of the ETS domain that are conserved across this family of transcription factors.

Localization of aggregating proteins in bacteria depends on the rate of addition.

Many proteins are observed to localize to specific subcellular regions within bacteria. Recent experiments have shown that proteins that have self-interactions that lead them to aggregate tend to localize to the poles. Theoretical modeling of the localization of aggregating protein within bacterial cell geometries shows that aggregates can spontaneously localize to the pole due to nucleoid occlusion. The resulting polar localization, whether it be to a single pole or to both was shown to depend on the rate of protein addition. Motivated by these predictions we selected a set of genes from Escherichia coli, whose protein products have been reported to localize when tagged with green fluorescent protein (GFP), and explored the dynamics of their localization. We induced protein expression from each gene at different rates and found that in all cases unipolar patterning is favored at low rates of expression whereas bipolar is favored at higher rates of expression. Our findings are consistent with the predictions of the model, suggesting that localization may be due to aggregation plus nucleoid occlusion. When we expressed GFP by itself under the same conditions, no localization was observed. These experiments highlight the potential importance of protein aggregation, nucleoid occlusion and rate of protein expression in driving polar localization of functional proteins in bacteria.