Biochemical and Biophysical Characterisation of Transcription Regulators Associated with Myosin VI

Myosin VI (MVI) is implicated in many biological processes including, cell migration, endocytosis and transcription. MVI achieves its plethora of cellular functions owing to its association with multiple binding partners. Here, two proteins associated with MVI - nuclear dot protein 52 (NDP52), a putative transcription regulator, and the established transcription factor, oestrogen receptor (ERα) - are biochemically and biophysically characterised to further elucidate the role of this motor protein in transcription. Optimisation of expression and purification conditions has enabled the successful purification of full-length ERα using BL21 E.coli. According to current literature, this is the first time this has been reported. This will set the foundation for future purification optimisation required for binding assays between MVI and ERα. NDP52 has only recently been characterised as a putative transcription co-activator, where it was shown to promote RNA polymerase II (RNAPII) transcription by relieving the auto-inhibition of MVI. Here, novel insights into the biochemical and biophysical properties of NDP52 are presented, including DNA-binding characteristics of different structural domains using fluorescence-based binding assays. Expectedly, C- terminal DNA-binding motifs likely confer binding to double-stranded DNA (dsDNA) in vivo. Small-angle X-ray scattering (SAXS) data for full-length NDP52 is also presented, providing important structural information, given that no full-length crystal structure for NDP52 is currently available. Methods such as total internal reflection fluorescence (TIRF) microscopy, size-exclusion chromatography-multi- angle light scattering (SEC-MALS) and cryogenic super-resolution microscopy have also provided an understanding of the oligomeric state of NDP52 and its tertiary structure. Together, this evidence suggests that NDP52 functions as a parallel homodimer in vivo, which provides important structure-function insights into NDP52's role in transcriptional regulation. For instance, the parallel arrangement of the NDP52 dimer may facilitate tight binding to dsDNA, where each monomer binds either side of DNA, effectively clamping it