title: Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS.
abstract: The compositional and conformational changes during catalytic activation of the spliceosome promoted by the DEAH box ATPase Prp2 are only poorly understood. Here, we show by dual-color fluorescence cross-correlation spectroscopy (dcFCCS) that the binding affinity of several proteins is significantly changed during the Prp2-mediated transition of precatalytic B(act) spliceosomes to catalytically activated B* spliceosomes from Saccharomyces cerevisiae. During this step, several proteins, including the zinc-finger protein Cwc24, are quantitatively displaced from the B* complex. Consistent with this, we show that Cwc24 is required for step 1 but not for catalysis per se. The U2-associated SF3a and SF3b proteins Prp11 and Cus1 remain bound to the B* spliceosome under near-physiological conditions, but their binding is reduced at high salt. Conversely, high-affinity binding sites are created for Yju2 and Cwc25 during catalytic activation, consistent with their requirement for step 1 catalysis. Our results suggest high cooperativity of multiple Prp2-mediated structural rearrangements at the spliceosome's catalytic core. Moreover, dcFCCS represents a powerful tool ideally suited to study quantitatively spliceosomal protein dynamics in equilibrium.
authors.affiliation: Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ohrt
authors.forename: Mira
authors.initials: M
authors.lastname: Prior
authors.forename: Julia
authors.initials: J
authors.lastname: Dannenberg
authors.forename: Peter
authors.initials: P
authors.lastname: Odenwälder
authors.forename: Olexandr
authors.initials: O
authors.lastname: Dybkov
authors.forename: Nicolas
authors.initials: N
authors.lastname: Rasche
authors.forename: Jana
authors.initials: J
authors.lastname: Schmitzová
authors.forename: Ingo
authors.initials: I
authors.lastname: Gregor
authors.forename: Patrizia
authors.initials: P
authors.lastname: Fabrizio
authors.forename: Jörg
authors.initials: J
authors.lastname: Enderlein
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D020134:Catalytic Domain; D053487:DEAD-box RNA Helicases / Q000737:chemistry*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods; D017381:Spliceosomes / Q000737:chemistry*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; C066490:PRP2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases