tRNA structure and ribosomal function. I. tRNA nucleotide 27-43 mutations enhance first position wobble

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Transfer RNA (tRNA) su7 G36, a derivative of tRNA(Trp), possesses a 3'GUC anticodon complementary to the glutamine codon CAG.
• This specific tRNA is known to suppress UAG (amber) termination codons through a G-U wobble at the first codon position, a normally disallowed interaction.

Purpose of the Study:

• To investigate the role of the anticodon arm, particularly nucleotides 27-43, in regulating G-U wobble and tRNA function.
• To quantify the impact of mutations on amber suppression and ribosomal function using a lacZ reporter system.

Main Methods:

• Saturating mutagenesis of the anticodon arm of su7 G36 tRNA.
• Screening for enhanced UAG suppression using a lacZ reporter assay.
• Measurement of amber suppression, tRNA levels, and aminoacylation to calculate KUAG (an index of ribosomal function).
• Construction and comparison of all 16 permutations of nucleotides 27-43.

Main Results:

• Mutations in the anticodon helix, especially at nucleotides 27-43, significantly increased the G-U wobble at the first codon position by up to 40-fold.
• The parental tRNA structure appears optimized to minimize this type of miscoding.
• A conformational change involving nucleotides 27-43 differentially affects tRNA coding function, aminoacylation, and tRNA levels.
• Efficient wobble showed a strong inverse correlation with aminoacylation and tRNA levels.

Conclusions:

• Normal tRNA structure, particularly the top base-pair of the anticodon helix (nt 27-43), prevents amino acid misincorporation due to aberrant first position wobble.
• Mutations at nt 27-43 alter tRNA conformation, impacting its function on the ribosome.
• The observed inverse correlation suggests selection for tRNAs with optimal aminoacylation and stability, potentially limiting wobble in natural systems.