Ribosome-induced mRNA pseudoknot interactions visualized by DMS MaP-Seq.

Area of Science:

• Molecular Biology
• Structural Biology
• Virology

Background:

• RNA dynamics are crucial for biological functions, with the ribosome interacting with diverse RNA structures during translation.
• RNA pseudoknots are known to impede ribosomal reading frame maintenance.
• Understanding pseudoknot-ribosome interactions is key to deciphering translation fidelity.

Purpose of the Study:

• To investigate the conformational changes of RNA pseudoknots induced by the ribosome.
• To characterize the stability and unfolding mechanisms of the Sugarcane Yellow Leaf Virus pseudoknot (ScYLV_PK).
• To establish dimethyl sulfate probing with mutational profiling and sequencing (DMS MaP-Seq) as a tool for studying ribosome-RNA interactions.

Main Methods:

• Utilized dimethyl sulfate probing with mutational profiling and sequencing (DMS MaP-Seq) to analyze RNA structure.
• Applied DMS MaP-Seq to examine the unfolding of the Sugarcane Yellow Leaf Virus pseudoknot (ScYLV_PK) in the presence of the ribosome.
• Investigated the role of specific residues and ribosome occupancy (A/P-site) in pseudoknot stability.

Main Results:

• The helical junction of ScYLV_PK is stabilized by the ribosome, dependent on hydrogen bonding at residue 27.
• The ribosome destabilizes wildtype ScYLV_PK independently of A/P-site occupancy.
• DMS MaP-Seq precisely detected ribosome-induced RNA conformational changes.

Conclusions:

• DMS MaP-Seq is a sensitive method for detecting ribosome-mediated RNA structural dynamics.
• Specific structural motifs, including hydrogen bonding at residue 27, govern pseudoknot stability during translation.
• Ribosome interactions with pseudoknots involve complex stabilization and destabilization mechanisms.