Single-molecule force measurements show that r-proteins assist 23S rRNA co-transcriptional folding.

Mechanical overstretching of individual RNA-DNA hybrids is used as a novel in vitro assay to prepare a co-transcriptional RNA structure and study its interaction with proteins. Dual optical traps hold two microscopic beads linked by an RNA-DNA molecular construct that is designed such that the RNA strand progressively peels off and folds when trap-to-trap distance increases. Subsequent distance reduction leads to duplex reannealing, RNA structure and its interaction with proteins are probed by continuously measuring force during this peeling/reannealing cycle. Focusing on the early stage of E.coli large ribosomal subunit assembly (domains I-II of 23S rRNA and early-binding r-proteins uL4, uL13, bL20, uL22, and uL24), we find that these five r-proteins stabilize the 23S rRNA structure: this property is notably characterized in our experiments by the observation that full reannealing is less frequent when the r-proteins are present than when they are absent. Sites of reannealing blockage were derived from the single-molecule data and compared with known RNA structural elements and r-protein binding sites of the bacterial ribosome. Our results also show that the five early-binding r-proteins bind the 23S rRNA co-transcriptionally, corroborating the classical "assembly gradient" hypothesis.