A UV-induced mutation in neurospora that affects translational regulation in response to arginine.
The Neurospora crassa arg-2 gene encodes the small subunit of arginine-specific carbamoyl phosphate synthetase. The levels of arg-2 mRNA and mRNA translation are negatively regulated by arginine. An upstream open reading frame (uORF) in the transcript's 5' region has been implicated in arginine-specific control. An arg-2-hph fusion gene encoding hygromycin phosphotransferase conferred arginine-regulated resistance to hygromycin when introduced into N. crassa. We used an arg-2-hph strain to select for UV-induced mutants that grew in the presence of hygromycin and arginine, and we isolated 46 mutants that had either of two phenotypes. One phenotype indicated altered expression of both arg-2-hph and arg-2 genes; the other, altered expression of arg-2-hph but not arg-2. One of the latter mutations, which was genetically closely linked to arg-2-hph, was recovered from the 5' region of the arg-2-hph gene using PCR. Sequence analyses and transformation experiments revealed a mutation at uORF codon 12 (Asp to Asn) that abrogated negative regulation. Examination of the distribution of ribosomes on arg-2-hph transcripts showed that loss of regulation had a translational component, indicating the uORF sequence was important for Arg-specific translational control. Comparisons with other uORF5 suggest common elements in translational control mechanisms.
Freitag
M
M
Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science & Technology, Portland 97291-1000, USA.
Dighde
N
N
Sachs
M S
MS
D000595:Amino Acid Sequence; D001120:Arginine / Q000494:pharmacology*; D001483:Base Sequence; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; D017931:DNA Primers / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D017930:Genes, Reporter; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009492:Neurospora crassa / Q000187:drug effects* / Q000235:genetics* / Q000528:radiation effects; D016366:Open Reading Frames; D010641:Phenotype; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D017931:DNA Primers; D012331:RNA, Fungal; D012333:RNA, Messenger; D001120:Arginine; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D008025:Ligases; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; C064716:carbamoyl phosphate synthetase (arginine-specific)