Anticodon loop mutations perturb reading frame maintenance by the E site tRNA.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The ribosomal E site plays a crucial role in maintaining the translation reading frame.
• Mutations in transfer RNA (tRNA), particularly in the anticodon loop, can significantly impact translation fidelity.
• The amber suppressor tRNA, Su7, is a key molecule for studying these effects.

Purpose of the Study:

• To investigate the impact of mutations in the anticodon loop and stem of the Su7 tRNA on its ability to maintain the ribosomal reading frame in the E site.
• To assess the role of the "Hirsh" mutation in the D stem on frame maintenance.
• To determine the effect of the absence of ms(2)io(6)A nucleoside modifications at A37 on tRNA's frame-holding capacity in the E site.

Main Methods:

• Utilizing the Escherichia coli RF2 programmed frameshift site to quantify reading frame maintenance.
• Introducing saturation mutations in the anticodon loop of the Su7 tRNA.
• Introducing mutations in the anticodon stem and the D stem (Hirsh mutation) of the Su7 tRNA.
• Analyzing the effect of removing ms(2)io(6)A nucleoside modifications at A37.

Main Results:

• Most anticodon loop mutations increased frameshifting, suggesting decreased codon:anticodon stability, but the A site appears more sensitive to these structural changes.
• The Hirsh mutation unexpectedly increased frameshifting from the E site, potentially by enhancing tRNA's ability to interact with the A site.
• The absence of ms(2)io(6)A nucleoside modifications at A37 did not impair the tRNA's frame-holding ability in the E site, contrary to expectations based on its effects in other ribosomal sites.

Conclusions:

• The ribosomal E site exhibits distinct substrate requirements compared to the A and P sites.
• Anticodon loop structure is critical for tRNA function, but its impact on the E site's frame-holding capacity differs from its impact on other ribosomal sites.
• The Hirsh mutation's effect suggests a complex interplay between tRNA structure, ribosomal site interactions, and translational fidelity.