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CUG as a mutant start codon for cat-86 and xylE in Bacillus subtilis
N P Ambulos1, T Smith, W Mulbry
1Department of Biological Sciences, University of Maryland Baltimore County, Catonsville 21228.
Gene
|September 28, 1990
Summary
The study reveals that while UUG is the start codon for the cat-86 gene, AUG significantly boosts expression in Bacillus subtilis. CUG can function as a start codon in B. subtilis, though less efficiently, and its efficiency is context-dependent.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The cat-86 gene encodes chloramphenicol acetyltransferase (CAT), with a non-canonical UUG start codon.
- Standard start codons in related genes are typically AUG.
Purpose of the Study:
- To investigate the impact of altering the start codon of the cat-86 gene on its expression in Bacillus subtilis.
- To determine if CUG can function as a start codon in B. subtilis and under what conditions.
Main Methods:
- Site-directed mutagenesis was used to change the start codon of the cat-86 gene to AUG, GUG, and CUG.
- Expression levels were quantified by measuring CAT activity.
- N-terminal sequencing was performed on CAT protein from the CUG mutant.
- The xylE gene's start codon was changed to CUG, and its ribosome-binding site was optimized.
Main Results:
- Changing the cat-86 start codon from UUG to AUG increased expression by 36%.
- GUG and CUG as start codons decreased cat-86 expression to 65% and 30% of AUG levels, respectively.
- N-terminal sequencing confirmed CUG functions as a start codon, specifying methionine in B. subtilis.
- Optimizing the ribosome-binding site and spacing for xylE allowed CUG to function with 13% activity compared to AUG.
Conclusions:
- CUG can function as a start codon in B. subtilis, albeit with modest efficiency.
- Start codon efficiency is influenced by the specific gene and surrounding sequences.
- CUG is less efficient as a start codon in Escherichia coli compared to B. subtilis.