| dc.description.abstracteng | RNA helicases of the DEAH-box family fulfill essential functions in various aspects of RNA metabolism by reorganizing structured RNAs and RNPs through ATP-dependent motility in 3’ to 5’ direction along single-strands. Prp43 acts on several targets in pre-mRNA splicing and ribosome biogenesis. In the specific cellular context Prp43 is activated by G-patch factors which enhance its intrinsically low ATPase and RNA unwinding activity. To initiate unwinding, a single stranded RNA segment preceding the helicase target needs to be accommodated in a binding channel formed between the helicase core and the C-terminal domains. When Prp43 loads onto its substrate, the channel is thought to widen into a groove and fold around the RNA, but how the loading process is regulated by the substrates, ATP and RNA, and the gp binding partners remains unknown. While structural studies revealed that the gp motif connects the flexible C-terminal domains to the helicase core, the impact on RecA domain movement during catalysis, conformation of the binding channel and the unwinding of RNA substrates is unclear. In this thesis, a single-molecule approach was used to gain time resolved information on the effects of G-patch binding on the conformational dynamics of Prp43 and the interaction with its RNA substrate.
In a first project, a smFRET system reporting on the RecA domain movement of Prp43 gave insights in conformational dynamics during ATP hydrolysis and their modulation by interaction with Pfa1(gp). While the RecA domains adopt a closed conformation in the apo and nucleotide-bound states, addition of Pfa1(gp) induces an open state that is incompatible with nucleotide-binding and facilitates ADP release. Thereby, transitions from the weak (ADP) to the strong (apo) RNA binding state are accelerated. In a second smFRET system, labels were placed on the RNA to probe substrate binding and unwinding. In complex with Pfa1(gp), Prp43(ADP) switches between bound and unbound states instead of dissociating from the RNA, while the closure of RecA domains upon ATP binding generates sufficient force to separate proximal RNA structures. During ATP turnover, Pfa1(gp) accelerates the conformational cycling of the RecA domains. Thereby, processive movement along the substrate is enabled, as translocation becomes faster than drop-off from the RNA.
In a second project, a different smFRET system was used to study the conformational dynamics of the RNA binding channel. In the absence of binding partners, the channel alternates between open and closed conformations, but is preferentially closed. Binding of ATP and Pfa1(gp) shifts the equilibrium towards the open state, facilitating the accommodation of RNA. After the initial loading step, the channel remains firmly closed during continuous cycles of ATP hydrolysis, enabling processive translocation. In contrast, no efficient RNA loading takes place in absence of Pfa1(gp).
Combining observations from all systems revealed how G-patch partners regulate the interaction of Prp43 with its substrates ATP and RNA by modulating the motility of its RecA domains and RNA binding channel. This led to a mechanistic model of its motility cycle. | de |