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. [ABSTRACT FROM AUTHOR]