Mutations affecting excision of the intron from a eukaryotic dimeric tRNA precursor.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Transfer RNA (tRNA) genes are crucial for protein synthesis, and their structure and function are highly conserved.
• Opal suppressor tRNA genes, like sup9-e in Schizosaccharomyces pombe, play a role in decoding stop codons.
• Mutations in tRNA genes can lead to altered gene expression and processing, impacting cellular function.

Purpose of the Study:

• To determine the nucleotide sequences of the Schizosaccharomyces pombe opal suppressor serine tRNA gene (sup9-e) and its 12 non-suppressing mutants.
• To analyze the effects of these mutations on tRNA gene transcription and precursor tRNA processing in Saccharomyces cerevisiae.
• To elucidate the relationship between specific mutations and defects in tRNA function.

Main Methods:

• Nucleotide sequencing of wild-type and mutant tRNA genes.
• In vitro and in vivo expression analysis in Saccharomyces cerevisiae.
• Analysis of tRNA precursor splicing and processing.
• Investigation of transcription of adjacent tRNA genes.

Main Results:

• Sequence determination revealed mutations affecting the D-loop, intron, extra arm, and anticodon stem of the tRNA gene.
• Mutations were found to cause defects in tRNA gene transcription and precursor tRNA processing.
• Specific mutations impacted splicing efficiency and accuracy, with one mutation allowing alternative base pairing.
• A single base change (G----A19) in the tRNASer gene abolished transcription of both the tRNASer and adjacent tRNAMet genes.

Conclusions:

• Mutations in the sup9-e tRNA gene disrupt UGA suppression through impaired transcription and precursor processing.
• The D-loop, intron, and extra arm are critical for efficient and accurate tRNA splicing.
• A specific mutation suggests that the tRNAMet gene is processed from a dimeric precursor, highlighting complex gene organization and expression strategies.