The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Viral gene expression often relies on translational readthrough of stop codons.
• The mechanism of readthrough for Tobacco Mosaic Virus (TMV) UAG stop codons has been debated, with hypotheses including tRNA non-conventional base-pairing and tRNA hopping.
• Understanding this process is crucial for viral replication and gene expression strategies.

Purpose of the Study:

• To investigate the role of sequences flanking the leaky UAG stop codon in TMV translational readthrough.
• To determine the minimal sequence context required for readthrough.
• To differentiate between tRNA-mediated mechanisms and sequence context effects on stop codon bypass.

Main Methods:

• Utilized an in vivo assay in tobacco protoplasts coupling stop codon bypass to beta-glucuronidase (GUS) reporter gene expression.
• Analyzed GUS constructions with altered codons flanking the UAG stop codon.
• Performed single-base mutation analysis on flanking codons.

Main Results:

• Identified the minimal sequence for readthrough as UAG-CAA-UUA.
• Determined that 3' contexts of the form CAR-YYA confer leakiness and permit readthrough of UAG, UAA, and UGA stop codons.
• Demonstrated a significant role for the 3' sequence context in readthrough, independent of tRNA hopping or solely anticodon-codon interactions.

Conclusions:

• The 3' sequence context plays a major role in translational readthrough of stop codons.
• The findings do not support models relying exclusively on anticodon-codon interactions or tRNA hopping for TMV UAG stop codon bypass.
• A unique sequence element in the 3' context appears to regulate translation termination.