Kinetics of ribosomal pausing during programmed -1 translational frameshifting

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Programmed -1 ribosomal frameshifting is crucial for translating essential viral RNA in Saccharomyces cerevisiae.
• This process relies on a specific slippery site and a downstream pseudoknot.
• Previous studies indicated ribosomes pause at the slippery site during frameshifting.

Purpose of the Study:

• To investigate the kinetics of ribosomal pausing and frameshifting at the slippery site.
• To quantify the rates of synthesis, pause times, and frameshifting efficiency.
• To elucidate the mechanisms by which ribosomes navigate the frameshift site.

Main Methods:

• In vitro translation assays were employed.
• Quantitative heelprinting was used to detect translational intermediates.
• Ribosomal pause times and frameshifting fractions were estimated.

Main Results:

• Approximately 10% of ribosomes paused at the slippery site in vitro.
• About 60% of paused ribosomes proceeded in the -1 frame.
• Ribosomes frameshifting into the -1 frame exhibited pause times significantly longer than peptide bond synthesis.
• Altering translational initiation rates affected frameshifting in vivo.

Conclusions:

• Three distinct mechanisms govern ribosome passage at the frameshift site.
• Only one of these mechanisms leads to successful -1 ribosomal frameshifting.
• The rate of translational initiation influences frameshifting efficiency in vivo.