UGA can be decoded as tryptophan at low efficiency in Bacillus subtilis.

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• The cat-86 gene encodes chloramphenicol acetyltransferase, conferring resistance to the antibiotic chloramphenicol.
• Codon usage and termination signals play critical roles in gene expression regulation.
• Understanding how altered codons affect gene function is crucial for genetic engineering.

Purpose of the Study:

• To investigate the functional consequences of replacing specific cat-86 codons with UGA (a stop codon).
• To determine the impact of these mutations on chloramphenicol resistance in Bacillus subtilis.
• To analyze the translational decoding of UGA codons in wild-type and suppressor-positive strains.

Main Methods:

• Site-directed mutagenesis was used to introduce UGA codons at positions 7 and 144 of the cat-86 gene.
• Assays for chloramphenicol resistance were performed on wild-type and mutant Bacillus subtilis strains.
• N-terminal sequencing was employed to identify the amino acid incorporated at the mutated UGA codon.

Main Results:

• Replacement of cat-86 codons 7 or 144 with UGA conferred chloramphenicol resistance in wild-type Bacillus subtilis.
• UAA replacements at the same codons resulted in sensitivity in wild-type strains but resistance in suppressor-positive strains.
• N-terminal sequencing revealed that UGA at codon 7 was inefficiently decoded as tryptophan (approx. 6% efficiency) in wild-type cells.

Conclusions:

• The UGA codon can be contextually decoded as an amino acid (tryptophan) in Bacillus subtilis, leading to functional protein production.
• This context-dependent decoding of UGA codons offers a potential mechanism for modulating gene expression and protein function.
• The findings have implications for understanding translational control and developing novel genetic tools.