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Structural and functional studies on two ATP-driven nucleic acid machines from viruses

Ge, Mengyu (2019) Structural and functional studies on two ATP-driven nucleic acid machines from viruses. PhD thesis, University of York.

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Abstract

ATP-driven motors exist globally in almost all prokaryotic and eukaryotic organisms. The motors in viruses are essential for viral replication and assembly. In this thesis, two ATP-driven nucleic acid motors from viruses were studied: a DNA packaging ATPase present in Thermus thermophilus phage phiKo and an NS3 helicase from Zika virus. The production and purification of the DNA packaging ATPase (gp14) from the phage phiKo was investigated. The purified gp14 protein was crystallised for determination of the native structure as well as the complexes with several non-hydrolysable ATP analogues, representing a precise picture of the ATP hydrolysis cycle. Structural analysis reveals that gp14 possesses an FtsK-like fold and likely translocates DNA similarly to other FtsK/HerA superfamily translocases. Moreover, an additional catalytic sensor residue from the adjacent subunit is required for ATP hydrolysis and protein assembly. The appropriate assembly of gp14 may be achieved by co-assembling with an auxiliary protein component into a higher-order hetero-oligomer complex. Further characterisations, including thermal unfolding, ATPase activity and DNA binding assays elucidate that gp14 is thermodynamically stable, but its ATPase activity decreases over time. The purified Zika virus NS3 helicase (NS3h) was crystallised in its apo form and as complexes with several nucleotides, including pre-hydrolysis substrate, transition state, and post-hydrolysis product analogues, as well as in a complex with a single-stranded RNA (ssRNA) segment containing a 5’ phosphate. The structural observations illustrate how a catalytically important motif mediates the coupling of ATP hydrolysis with RNA translocation. Due to the transition state analogue ADP-MgF3(H2O)- not being observed before, this complex structure was subjected to careful examination by crystallography and 19F NMR. Further functional characterisations, including protein stability, RNA binding and ATPase activity assays, demonstrate this NS3 helicase possesses weak RNA-binding mode and its ATPase activity can be stimulated by ssRNA.

Item Type: Thesis (PhD)
Academic Units: The University of York > Chemistry (York)
Depositing User: Mengyu Ge
Date Deposited: 16 Dec 2019 13:08
Last Modified: 16 Dec 2019 13:08
URI: http://etheses.whiterose.ac.uk/id/eprint/25540

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