A yeast knockout strain to discriminate between active and inactive tRNA molecules

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Transfer RNA (tRNA) molecules are crucial for protein synthesis, carrying specific amino acids to the ribosome.
• The structure and function of tRNA(Arg) (tRNA for arginine) are vital for cellular processes, but essential residues remain to be fully elucidated.
• Aminoacyl-tRNA synthetases (aaRS) are responsible for charging tRNAs with their cognate amino acids, a process critical for translation fidelity.

Purpose of the Study:

• To identify essential residues in yeast tRNA(Arg) required for its function.
• To investigate the roles of specific tRNA(Arg) structural domains, particularly the D and anticodon loops, in aminoacylation and translation.
• To compare the identity elements of yeast tRNA(Arg) with those found in other organisms, such as Escherichia coli.

Main Methods:

• Development of a yeast genetic screening system to select for inactive tRNA(Arg) variants.
• Plasmid shuffling technique to isolate and select for tRNA mutants.
• Northern blotting to analyze the abundance of tRNA(Arg) variants.
• Mutational analysis focusing on key tRNA structural regions.

Main Results:

• Most inactivating mutations occurred in residues involved in tertiary interactions, significantly affecting tRNA(Arg) abundance.
• Mutations in the anticodon loop, while not affecting transcription or aminoacylation, likely impaired interactions with the translation machinery.
• Individual mutations at residues 16, 20, and 38, known to interact with aminoacyl-tRNA synthetase, did not cause lethality.
• Steady-state levels of aminoacylated U20A and U20G variants decreased significantly in vivo, suggesting residue 20 has limited importance in yeast compared to E. coli.

Conclusions:

• Yeast tRNA(Arg) function relies heavily on residues involved in tertiary structure, impacting tRNA stability and abundance.
• The anticodon loop plays a role in translation beyond decoding, potentially through interactions with ribosomal components.
• Residue 20 of tRNA(Arg) is not a major identity element in yeast, contrasting with its critical role in Escherichia coli, highlighting species-specific differences in tRNA recognition.