The spliceosome is a dynamic ribonucleoprotein (RNP) machine that catalyzes the removal of introns in 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 branchpoint (BP) of pre-mRNA in affinity-purified spliceosomes stalled by a mutation in the DExD/H-box helicase Prp2 immediately prior to 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 then strongly biases this equilibrium towards 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.
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