Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS
- Thomas Ohrt1,3,
- Mira Prior2,3,
- Julia Dannenberg1,
- Peter Odenwälder1,
- Olexandr Dybkov1,
- Nicolas Rasche1,
- Jana Schmitzová1,
- Ingo Gregor2,
- Patrizia Fabrizio1,
- Jörg Enderlein2,4 and
- Reinhard Lührmann1,4
- 1Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany
- 2III. Physikalisches Institut (Biophysik), University of Göttingen, D-37077 Göttingen, Germany
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↵3 These authors contributed equally to this work.
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 Bact 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.
Keywords
Footnotes
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↵4 Corresponding authors.
E-mail reinhard.luehrmann{at}mpi-bpc.mpg.de.
E-mail joerg.enderlein{at}physik3.gwdg.de.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.033316.112.
- Received March 19, 2012.
- Accepted March 21, 2012.