Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing.
The spliceosome is a dynamic ribonucleoprotein (RNP) machine that catalyzes the removal of introns during the two transesterification steps of eukaryotic pre-mRNA splicing. Here we used single-molecule fluorescence resonance energy transfer to monitor the distance of the 5' splice site (5' SS) and branch point (BP) of pre-mRNA in affinity-purified spliceosomes stalled by a mutation in the DExD/H-box helicase Prp2 immediately before the first splicing step. Addition of recombinant Prp2 together with NTP and protein cofactor Spp2 rearranges the spliceosome-substrate complex to reversibly explore conformations with proximal 5' SS and BP that accommodate chemistry. Addition of Cwc25, a small heat-stable splicing factor, then strongly biases this equilibrium toward the proximal conformation, promoting efficient first-step splicing. The spliceosome thus functions as a biased Brownian ratchet machine where a helicase unlocks thermal fluctuations subsequently rectified by a cofactor 'pawl', a principle possibly widespread among the many helicase-driven RNPs.
Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan, USA.
Ramya
R
Krishnan
Mario R
MR
Blanco
Matthew L
ML
Kahlscheuer
John
J
Abelson
Christine
C
Guthrie
Nils G
NG
Walter
D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D012322:RNA Precursors / Q000378:metabolism*; D022821:RNA Splice Sites; D012326:RNA Splicing / Q000502:physiology*; D000072260:RNA Splicing Factors; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017381:Spliceosomes / Q000235:genetics / Q000502:physiology; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism
C544745:Cwc25 protein, S cerevisiae; D012322:RNA Precursors; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; C096994:SPP2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C501306:Ubc4 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; C066490:PRP2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases