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Early Steps of Ribosome Assembly Studied by Single Molecule Force Measurements

Abstract : Double-stranded DNA and RNA (dsDNA, dsRNA) undergo overstretching transitions at forces around 60 pN. We perform force measurements using a dual-beam optical trap that holds two beads linked by a single molecule. One strand of the duplex is attached at both extremities to the beads, while the other strand is attached only at one extremity. Four different cases are compared: dsDNA, dsRNA, RNA-DNA hybrid with DNA under tension, and DNA-RNA hybrid with RNA under tension. Overstretching occurs for all four duplexes. The most remarkable difference is that dsRNA exhibits a smooth plateau, while the other duplexes show saw-tooth patterns. We find that dsRNA overstretches by a different mechanism and explain why this property could help RNA structures to assemble and play their biological roles.Overstretching the RNA-DNA hybrid progressively liberates an RNA strand. Structures formed within this nascent RNA are seen in the force signal upon re-annealing. For the first time to our knowledge, we thus mimic and study co-transcriptional RNA folding in an in-vitro assay. Focusing on the early stage of E.coli large ribosomal subunit assembly (domains I-II of 23S rRNA and r-proteins L4, L13, L20, L22, L24), partial re-annealing is observed more frequently with r-proteins than without. Our results indicate that the five early- binding r-proteins act as folding helpers well before the entire 23S RNA is transcribed.
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Lena Melkonyan. Early Steps of Ribosome Assembly Studied by Single Molecule Force Measurements. Physics [physics]. Université Paris sciences et lettres, 2018. English. ⟨NNT : 2018PSLET026⟩. ⟨tel-02333812⟩



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