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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. 

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. 

Abstract Ligand-induced endocytosis of the immune receptor FLAGELLIN SENSING2 (FLS2) is critical for maintaining its proper abundance in the plasma membrane (PM) to initiate and subsequently down regulate cellular immune responses to bacterial flagellin or flg22-peptide. The molecular components governing PM abundance of FLS2, however, remain mostly unknown. Here, we identified Arabidopsis (Arabidopsis thaliana) DYNAMIN-RELATED PROTEIN1A (DRP1A), a member of a plant-specific family of large dynamin GTPases, as a critical contributor to ligand-induced endocytosis of FLS2 and its physiological roles in flg22-signaling and immunity against Pseudomonas syringae pv. tomato DC3000 bacteria in leaves. Notably, drp1a single mutants displayed similar flg22-defects as those previously reported for mutants in another dynamin-related protein, DRP2B, that was previously shown to colocalize with DRP1A. Our study also uncovered synergistic roles of DRP1A and DRP2B in plant growth and development as drp1a drp2b double mutants exhibited severely stunted roots and cotyledons, as well as defective cell shape, cytokinesis, and seedling lethality. Furthermore, drp1a drp2b double mutants hyperaccumulated FLS2 in the PM prior to flg22-treatment and exhibited a block in ligand-induced endocytosis of FLS2, indicating combinatorial roles for DRP1A and DRP1B in governing PM abundance of FLS2. However, the increased steady-state PM accumulation of FLS2 in drp1a drp2b double mutants did not result in increased flg22 responses. We propose that DRP1A and DRP2B are important for the regulation of PM-associated levels of FLS2 necessary to attain signaling competency to initiate distinct flg22 responses, potentially through modulating the lipid environment in defined PM domains.