Effect of base sequence on in vitro protein-chain termination

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Nonsense codons signal the end of protein synthesis.
• The efficiency of translation termination is crucial for cellular function.
• Previous studies suggested flanking sequences might influence termination.

Purpose of the Study:

• To investigate the role of nucleotide sequences surrounding the UAA stop codon in prokaryotic translation termination.
• To determine how the nature and number of bases adjacent to UAA affect termination factor RF-1 activity.

Main Methods:

• Purification of termination factor RF-1.
• Synthesis of oligoribonucleotides with varying sequences around the UAA codon.
• Analysis of oligomer conformation using NMR spectroscopy.
• Assay of RF-1-dependent termination activity in vitro by measuring peptide analogue release from ribosomes.

Main Results:

• Oligomers with specific sequences surrounding UAA, such as AUGUAA, stimulated RF-1-mediated release.
• UAAUAA was significantly less effective (5-fold) in stimulating release compared to UAA alone or other variants.
• The conformation of UAA (loosely stacked vs. strongly stacked) correlated with termination efficiency.

Conclusions:

• Recognition of the UAA stop codon by RF-1 is influenced by adjacent nucleotide sequences.
• A loosely stacked conformation of UAA favors efficient termination.
• Strong base stacking in flanking regions can hinder translation termination.