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Proper codon/anticodon pairing within the ribosome necessitates linearity of the transcript. Any structures formed within a messenger RNA (mRNA) must be unwound before the respective codon is interpreted. Linearity, however, is not always the norm; some intricate structures within mRNA are able to exert unique ribosome/mRNA interactions to regulate translation. Intrinsic kinetic and thermal stability in many of these structures are efficient in slowing translation causing pausing of the ribosome. Altered translation kinetics arising from atypical interactions have been shown to affect intersubunit rotation. Here, we employ single-molecule Förster resonance energy transfer (smFRET) to observe changes in intersubunit rotation of the ribosome as it approaches downstream structured nucleic acid. The emergence of the hyperrotated state is critically dependent on the distance between downstream structure and the ribosome, suggesting interactions with the helicase center are allosterically coupled to intersubunit rotation. Further, molecular dynamics (MD) simulations were performed to determine ribosomal protein/mRNA interactions that may play a pivotal role in helicase activity and ultimately unwinding of downstream structure.
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Ribosome-induced mRNA pseudoknot interactions visualized by DMS MaP-Seq
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 (ScYLVPK). Notably, we find that the helical junction is stabilized in the presence of the ribosome and is contingent upon hydrogen bonding at the 27thresidue of ScYLVPK. Additionally, it is demonstrated that the ribosome destabilizes wildtype ScYLVPKin 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.
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- Award ID(s):
- 2122902
- PAR ID:
- 10658977
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Posted Content:
- https://doi.org/10.1101/2024.12.07.627321
