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 E. coli, whose protein products have been reported to localize when tagged with 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

Dissecting the biophysical properties and DNA-binding specificity of the ETV6 transcription factor

Transcription factors bind to specific DNA sequences and regulate the expression of associated genes. In this thesis, I used several biophysical methods to investigate the structure and DNA-binding specificity profile of the eukaryotic transcription factor ETV6. In chapter 2 I investigated the mechanisms by which ETV6 selectively binds its cognate DNA targets. ETV6 prefers DNAs containing a core GGAA motif, unlike other family members where GGA(A/T) is accepted. This specificity toward the fourth adenine is mediated by a single histidine, His396, contrasted with a tyrosine in other family members. Through NMR-monitored pH titrations, I found His396 adopts the neutral Nε2H tautomeric state when ETV6 is both free and DNA-bound. Both structural and surface plasmon resonance binding studies revealed the mutation of His396 to a tyrosine increased its affinity for GGAT-containing DNA, while not diminishing its affinity for DNAs with a GGAA core. Thus I propose that His396 does not serve to enable binding of DNA containing a GGAA core, but rather to disfavour association toward DNAs containing bases other than an adenine at this fourth position. This thereby restricts the transcriptional profile of ETV6 relative to other ETS factors. In chapter 3 I investigated conformational flexibility within the DNA-binding domain of ETV6. All ETS family proteins share a structurally conserved ETS domain, allowing association toward many redundant DNA sequences. These proteins can also recognize subtly different DNA regions to enable transcriptional specificity. I proposed a contributing factor toward the profile of DNAs each family member recognized was domain flexibility. Testing this, I introduced cavity-forming mutations in the ETV6 ETS domain, resulting in increase flexibility determined through relaxation-dispersion NMR experiments. Then, using microarrays, I showed the increase in flexibility weakened selection of near-cognate DNAs, and thereby increased the specificity for cognate DNAs. These flexibility changes predominantly affected selection of DNA bases contacted solely via their phosphodiester backbone, indicating a link between protein flexibility and specificity for the sequence-dependent shape of DNA. Collectively my thesis uncovered many biophysical properties of the ETV6 ETS domain and illustrated how these features contribute to its DNA binding specificity at a molecular level.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat