Systematic alterations in the anticodon arm make tRNA(Glu)-Suoc a more efficient suppressor

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Ochre suppressors are transfer RNAs (tRNAs) that recognize and translate stop codons.
• The tRNA(Glu)-Suoc is an inefficient ochre suppressor, limiting its utility in genetic studies.
• Normal tRNAs that translate Uxx codons possess specific structural features in their anticodon arm.

Purpose of the Study:

• To engineer more efficient ochre suppressor tRNAs through site-specific mutagenesis.
• To investigate the relationship between anticodon arm structure and suppressor efficiency.
• To identify key nucleotide alterations responsible for enhanced tRNA suppressor function.

Main Methods:

• Site-specific mutagenesis was employed to create variants of tRNA(Glu)-Suoc.
• Structural modifications focused on extending the anticodon arm region.
• Suppressor efficiency was assessed in a relevant biological context.

Main Results:

• Five more efficient variants of tRNA(Glu)-Suoc were successfully constructed.
• Suppressor efficiency correlated positively with the similarity of the modified anticodon arm to that of normal Uxx-translating tRNAs.
• One variant demonstrated a 230-fold increase in suppressor efficiency compared to the parental tRNA.
• Two variants exhibited altered responses to messenger RNA context, suggesting context-dependent mutations.

Conclusions:

• Extending the anticodon arm and increasing its similarity to normal tRNAs is an effective strategy for enhancing suppressor efficiency.
• Nucleotide alterations in both the helix and loop of the anticodon arm significantly impact efficiency.
• The study identified highly efficient suppressor variants and revealed context-dependent mutational effects.