Neurospora crassa supersuppressor mutants are amber codon-specific.
Neurospora crassa has 10 mapped supersuppressor (ssu) genes. In vivo studies indicate that they suppress amber (UAG) premature termination mutations but the spectrum of their functions remains to be elucidated. We examined seven ssu strains (ssu-1, -2, -3, -4, -5, -9, and -10) using cell-free translation extracts. We tested suppression by requiring it to produce firefly luciferase from a reading frame containing premature UAA, UGA, or UAG terminators. All mutants except ssu-3 suppressed UAG codons. Maximal UAG suppression ranged from 15% to 30% relative to controls containing sense codons at the corresponding position. Production from constructs containing UAA or UGA was 1-2%, similar to levels observed with all nonsense codons in wild-type and ssu-3 extracts. UAG suppression was also seen using [35S]Met to radiolabel polypeptides. Suppression enabled ribosomes to continue translation elongation as determined using the toeprint assay. tRNA from supersuppressors showed suppressor activity when added to wild-type extracts. Thus, these supersuppressors produce amber suppressor tRNA.
Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health and Science University, Beaverton, OR 97006-8921, USA.
Peng
P
Fang
Cheng
C
Wu
Matthew S
MS
Sachs
D000595:Amino Acid Sequence; D001483:Base Sequence; D015825:Chromosomes, Fungal; D003062:Codon / Q000235:genetics; D003433:Crosses, Genetic; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics*; D012331:RNA, Fungal; D012333:RNA, Messenger; D012343:RNA, Transfer / Q000235:genetics*; D013489:Suppression, Genetic; D013728:Terminator Regions, Genetic
D003062:Codon; D012331:RNA, Fungal; D012333:RNA, Messenger; D012343:RNA, Transfer