Show simple item record

Biophysical and Crystallographic Characterization of Spliceosomal DExD/H-box ATPases

dc.contributor.advisorFicner, Ralf Prof. Dr.
dc.contributor.authorHamann, Florian
dc.titleBiophysical and Crystallographic Characterization of Spliceosomal DExD/H-box ATPasesde
dc.contributor.refereeFicner, Ralf Prof. Dr.
dc.description.abstractengDEAH-box ATPases play a vital role in the activation, catalytic and disassembly steps of the spliceosome. They are key for the orchestration of conformational and compositional rearrangements during the splicing cycle. It has long been proposed that they do this by targeting double-stranded RNA networks and unwinding duplexes. However, recent structural as well as biochemical studies suggest that they rather act on single-stranded RNAs at the spliceosomal peripheries and thereby remotely remodel the spliceosome. While mainly structural approaches have tried to unravel the translocation mechanism of spliceosomal DEAH-box ATPases, only initial insights into the conformational dynamics of this family could be gained. It has been proven that the analysis of different ligand-bound states has been key to unveil domain movements and adenosine nucleotide-free states of spliceosomal DEAH-box ATPases still remained elusive at the beginning of this thesis. The determination of the apo structure of the Chaetomium thermophilum Prp22 revealed an intrinsic mobility of the RecA2 domain that allows open conformations of the helicase core previously undescribed. A bound RNA to the adenosine nucleotide-free Prp22 stabilizes the RecA2 domain into a defined open conformation of the helicase core. Compared to the RNA- and ATP-bound state of Prp43 with a closed helicase core conformation, the adenosine nucleotide-free state is able to accommodate an additional RNA nucleotide in the binding tunnel. A continuous toggling between closed and open conformations of the helicase core, enables DEAH-box ATPases to translocate along an ssRNA with a step-size of one RNA nucleotide per hydrolyzed ATP. In order to synchronize these domain movements, a serine in the conserved sequence motif V is able to sense the catalytic state and accordingly position the RecA2 domain. A further goal of this thesis was to study the discrepant way of functioning of Prp2. Prp2 plays a special role among spliceosomal DEAH-box ATPases as it is the only one not showing any in vitro unwinding capability. Comparing the ADP-BeF3-- and RNA-bound structure of Chaetomium thermophilum Prp2 with a Prp43 structure in the same catalytic state, a loop in the C-terminal domains could be identified that ensures a divergent way of interaction with the RNA in Prp2. An insertion in this loop in Prp2 threads the RNA differently through the binding tunnel and might play a role in impeding Prp2 from being an unwindase. Additionally, complex structures of a Prp2-Spp2 G-patch complex could be solved and provide the first glimpse of a G-patch domain bound to its target protein. The structures show that the N-terminal part of the G-patch stably binds the winged-helix domain of Prp2, while the C-terminal end binds the RecA2 domain with two alternative conformations. Together with a flexible linker connecting these two parts, the G-patch exhibits an increased conformational flexibility that enables it to adapt to the movements of the RecA2 domain during
dc.contributor.coRefereeLührmann, Reinhard Prof. Dr.
dc.contributor.thirdRefereeStark, Holger Prof. Dr.
dc.subject.engRNA helicasede
dc.subject.engDEAH-box ATPasede
dc.subject.engRNA translocationde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

Files in this item


This item appears in the following Collection(s)

Show simple item record