Study of Interactions Between the mRNA Pseudoknot and the Ribosome with Single-Molecule FRET

Pseudoknots are important and complex RNA structures. They play critical roles in many biological processes, such as RNA replication, transcription and translation. Several viruses use a pseudoknot structure to induce programmed ribosomal frameshifting during translation. Previous research used an H-type pseudoknot derived from the human telomerase RNA (DU177) as a model system, which has two helical stems connected by two single-stranded loops. DU177 can function as a -1 ribosomal frameshifting stimulator when it is positioned downstream of a slippery sequence. In addition, mRNA structures must be unfolded to a single-stranded form to be translated. Previous research has revealed that the ribosome has intrinsic helicase activity during translation. However, the detailed molecular mechanism of how a pseudoknot is unwound by the ribosome remains unclear. In this study, we use single-molecule fluorescence resonance energy transfer (smFRET) to elucidate how a pseudoknot is unwound by the ribosome. Our results show that when a partial pseudoknot sequence enters the ribosome during translation, the pseudoknot structure is distorted by the ribosome. The structural distortion may play a role in inducing the ribosome to undergo frameshifting. However, the ribosome can overcome the steric barrier to unwind the first stem of the pseudoknot completely when translation proceeds further. According to our results, we propose that, due to the presence of several base triples that stabilize the whole RNA structure, the folding of the pseudoknot may be maintained even if a partial sequence enters the ribosome.