Identifying the RNA Kinetic Traps in Ribosome Assembly

Abstract

mutant derivatives are investigated with optical tweezers, steered molecular dynamics (SMD) simulations and single-molecule FRET (smFRET). With smFRET, we show that base triples at one end (formed by loop 1 and stem 2) of hTR-PK promote the formation of those at the distal end (formed by stem 1 and loop 2), and thereby limit the flexibility of the distal loop (loop 2). The coordination between base triples is therefore responsible for the single-step unfolding event of the structure. By contrast, smFRET experiments identify a compact intermediate structure before hTR-PK is completely disrupted by the ribosome. SMD simulations further reveal that as the hTR-PK loop 2 attaches to the positively charged residues of ribosomal protein S3, base triples facilitate formation of the unfolding intermediate of hTR-PK. When the base triples are disrupted by mutations (the delta-triple mutant), the compact unfolding intermediate can no longer be seen in SMD simulations and smFRET experiments (that is, the mutant exhibits single-step unfolding). The delta-triple mutation also results in a dramatic drop in FS efficiency from 50% to 0%. Our study demonstrates the importance of a base triple-stabilized unfolding intermediate in PK-induced FS.