mRNA pseudoknot structures can act as ribosomal roadblocks

Area of Science:

• Molecular Biology
• Virology
• Biophysics

Background:

• Viruses use programmed ribosomal frameshifting, often involving mRNA pseudoknots and slippery sequences, to regulate protein production ratios.
• While a link between pseudoknot mechanical strength and frameshifting efficiency exists, the underlying physical mechanisms require further elucidation.

Purpose of the Study:

• To investigate the relationship between mRNA pseudoknot structure, mechanical strength, and frameshifting efficiency.
• To explore the physical mechanisms governing ribosomal navigation through pseudoknot structures.

Main Methods:

• Synthesis of RNA sequences designed to form pseudoknot-like structures with varying predicted strengths.
• Analysis of frameshifting efficiency using protein expression studies.
• Two-dimensional gel electrophoresis of pulse-labeled proteins to assess ribosome progression.

Main Results:

• Contrary to expectations, the strongest predicted pseudoknot structures resulted in limited frameshifting.
• A significant portion of ribosomes were observed to frameshift but were subsequently halted by the pseudoknot structures, acting as roadblocks.
• Increased pseudoknot strength correlated with higher frameshifting efficiency and a more pronounced roadblocking effect.

Conclusions:

• mRNA pseudoknots can function as ribosomal roadblocks, impeding the translation of frameshifted ribosomes.
• Optimal production of full-length frameshifted protein requires balancing pseudoknot strength for both frameshifting and minimal roadblocking.
• Understanding ribosomal roadblocking is crucial for developing accurate hypotheses regarding frameshifting mechanisms.