Arginine-specific Regulation Mediated by the Neurospora crassa arg-2 Upstream Open Reading Frame in a Homologous, Cell-free in Vitro Translation System

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.