Related Experiment Videos
Neurospora crassa supersuppressor mutants are amber codon-specific
Peng Fang1, Cheng Wu, Matthew S Sachs
1Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health and Science University, Beaverton, OR 97006-8921, USA.
Fungal Genetics and Biology : FG & B
|July 24, 2002
Summary
Seven Neurospora crassa supersuppressor (ssu) strains were analyzed for their ability to suppress premature stop codons. Most ssu mutants efficiently suppressed amber (UAG) mutations, indicating they produce amber suppressor transfer RNA (tRNA).
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Neurospora crassa possesses ten mapped supersuppressor (ssu) genes.
- In vivo studies suggest these genes suppress amber (UAG) premature termination mutations, but their functional range is not fully understood.
Purpose of the Study:
- To investigate the functional spectrum of seven Neurospora crassa supersuppressor (ssu) strains (ssu-1, -2, -3, -4, -5, -9, and -10).
- To determine the specific type of premature termination codons suppressed by these ssu mutants.
Main Methods:
- Utilized cell-free translation extracts from seven ssu strains.
- Tested suppression efficiency by measuring firefly luciferase production from constructs with premature UAA, UGA, or UAG terminators.
- Employed toeprint assays to assess ribosome translation elongation.
- Analyzed transfer RNA (tRNA) from supersuppressor strains for suppressor activity in wild-type extracts.
Main Results:
- Six of the seven tested ssu strains (ssu-1, -2, -4, -5, -9, and -10) effectively suppressed UAG codons, with maximal suppression ranging from 15% to 30%.
- Suppression of UAA and UGA codons was minimal (1-2%), comparable to wild-type levels.
- Toeprint assays confirmed that suppression allows ribosomes to continue translation elongation.
- tRNA isolated from supersuppressor strains exhibited suppressor activity when added to wild-type extracts.
Conclusions:
- The studied supersuppressor strains of Neurospora crassa primarily produce amber suppressor tRNA.
- These findings clarify the specific function of several ssu genes in nonsense-codon suppression.