Increased ribosomal accuracy increases a programmed translational frameshift in Escherichia coli

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Programmed ribosomal frameshifting is a mechanism regulating gene expression.
• Ribosomal fidelity, influenced by factors like tRNA availability, can impact frameshifting efficiency.
• Previous studies suggested limited tRNA availability facilitates frameshifting at specific codons.

Purpose of the Study:

• To investigate the effect of increased ribosomal fidelity on programmed translational frameshifting.
• To examine the role of tRNA availability in modulating frameshifting efficiency at sense codons.
• To test the hypothesis that ribosome pause time at frameshift sites is a key determinant of frameshift efficiency.

Main Methods:

• Utilized modified release factor 2 (RF2) translational frameshift constructs with UGG (tryptophan) or CUG (leucine) codons.
• Employed wild-type and hyperaccurate streptomycin pseudo-dependent ribosomal hosts.
• Manipulated transfer RNA (tRNA) levels through plasmid-based gene induction.

Main Results:

• Hyperaccurate ribosomes significantly increased UGG-mediated frameshifting (>50%), contrary to expectations.
• Increased tRNA(Trp) levels reduced frameshifting in hyperaccurate hosts, restoring it to near wild-type levels (~7%).
• Increased accuracy alleles also increased UGG frameshifting and slowed translation rates, with effects minimal on CUG frameshifts.

Conclusions:

• Ribosome pause time at programmed frameshift sites is a critical factor influencing frameshift efficiency.
• Tighter ribosomal fidelity can paradoxically enhance frameshifting by increasing pause duration.
• tRNA availability directly modulates frameshift efficiency by altering ribosome pause times at specific codons.