An engineered class I transfer RNA with a class II tertiary fold.

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Transfer RNAs (tRNAs) exhibit distinct structural classes (I and II) crucial for protein synthesis.
• Escherichia coli tRNA(Gln)2 is a class I tRNA essential for glutamine incorporation.
• Understanding tRNA structural determinants is vital for deciphering gene expression mechanisms.

Purpose of the Study:

• To engineer the tertiary fold of Escherichia coli tRNA(Gln)2 into a class II structure.
• To investigate the structural requirements for maintaining tRNA stability and function after structural conversion.
• To explore the impact of altered tRNA tertiary structure on glutaminyl-tRNA synthetase recognition.

Main Methods:

• Structure-based engineering of Escherichia coli tRNA(Gln)2.
• Incorporation of the 20-nucleotide variable stem-loop from Thermus thermophilus tRNA(Ser).
• Analysis of tertiary structure stability and glutaminylation efficiency.

Main Results:

• Successful conversion of Escherichia coli tRNA(Gln)2 to a class II structure while preserving glutamine recognition.
• Enlargement of the D-loop was essential for maintaining tertiary structure stability.
• Rearrangement of a base triple in the D-stem was critical for efficient glutaminylation.
• Glutaminyl-tRNA synthetase demonstrated significant sensitivity to changes in tRNA tertiary structure.

Conclusions:

• The study provides novel insights into the structural determinants that differentiate class I and class II tRNA folds.
• Demonstrates the critical role of the D-loop and D-stem modifications in tRNA structural integrity and function.
• Highlights the high sensitivity of glutaminyl-tRNA synthetase to alterations in tRNA tertiary structure, impacting aminoacylation specificity.