Arginine-specific regulation mediated by the Neurospora crassa arg-2 upstream open reading frame in a homologous, cell-free in vitro translation system.
Translational control mediated by an upstream open reading frame (uORF) in the 5'-leader of the Neurospora crassa arg-2 mRNA was reconstituted in a homologous, cell-free in vitro translation system. A cell-free N. crassa system was developed that required the presence of cap and poly(A) on RNA for maximal translation and that was amino acid-dependent. The 24-codon arg-2 uORF, when placed in the 5'-leader region of capped and adenylated synthetic luciferase RNAs, conferred Arg-specific negative regulation in this system. Improving the uORF translation initiation context decreased luciferase production and only slightly increased the magnitude of Arg-specific regulation. Mutation of uORF Asp codon 12 to Asn, which eliminates Arg-specific regulation in vivo, eliminated regulation in vitro. Elimination of the uORF translation initiation codon also eliminated Arg-specific regulation. Arg-specific regulation in vitro appeared to be reversible. Control of RNA stability did not appear to be a primary component of Arg-specific regulation in vitro. Comparison of the effects of adding Arg to in vitro translation reactions with adding compounds related to Arg indicated that Arg-specific translational regulation was specific for L-arginine.
Wang
Z
Z
Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science & Technology, Portland 97291-1000, USA.
Sachs
M S
MS
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D002474:Cell-Free System; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000502:physiology*; D011061:Poly A / Q000235:genetics; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid
D012315:RNA Caps; D012333:RNA, Messenger; D011061:Poly A; D001120:Arginine