Arginine-specific regulation mediated by the Neurospora crassa arg-2 upstream open reading frame in a homologous, cell-free in vitro translation system

J Biol Chem. 1997 Jan 3;272(1):255-61. doi: 10.1074/jbc.272.1.255.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Arginine / metabolism*
  • Base Sequence
  • Cell-Free System
  • Gene Expression Regulation, Fungal
  • Molecular Sequence Data
  • Neurospora crassa / physiology*
  • Poly A / genetics
  • Protein Biosynthesis
  • RNA Caps / physiology
  • RNA, Messenger / metabolism
  • Regulatory Sequences, Nucleic Acid*

Substances

  • RNA Caps
  • RNA, Messenger
  • Poly A
  • Arginine

Associated data

  • GENBANK/J05512