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Abstract Examining the dynamicity of RNA structure has deepened our understanding of its vast biological functions. Perhaps the protein complex that encounters the most diverse landscape of RNA structure is the ribosome. In translation, the ribosome must linearize countless mRNA conformations for proper protein production. Some RNA structures, however, reliably make up sequences which hinder the ability of the ribosome to maintain its reading frame. The most well-studied of these structures is the RNA pseudoknot. Here, we present an approach utilizing dimethyl sulfate probing with mutational profiling and sequencing (DMS MaP-Seq) to precisely examine RNA unwinding. We employ the method to understand the unfolding of the Sugarcane Yellow Leaf Virus pseudoknot (ScYLV_PK). Notably, we find that the helical junction is stabilized in the presence of the ribosome and is contingent upon hydrogen bonding at the 27^thresidue of ScYLV_PK. Additionally, it is demonstrated that the ribosome destabilizes wildtype ScYLV_PKin a manner independent of A/P-site occupancy. Together, these results establish DMS MaP-Seq as a sensitive tool for detecting ribosome-induced RNA conformational changes and reveal specific structural motifs that govern pseudoknot stability during translation.