Role of an upstream open reading frame in mediating arginine-specific translational control in Neurospora crassa

• Published in: Journal of Bacteriology Vol. 178; no. 8; pp. 2172 - 2177
• Main Authors: Luo, Z. (Oregon Graduate Institute of Science and Technology, Portland, OR.), Sachs, M.S
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.04.1996
• Subjects: ADN RECOMBINADO; ADN RECOMBINE; ARGININA; ARGININE; Arginine - pharmacology; ARN MENSAJERO; ARN MESSAGER; Bacteriology; BETA GALACTOSIDASA; BETA GALACTOSIDASE; Biochemistry; biosynthesis; drug effects; E coli; enzymology; GENE; Gene Expression Regulation, Fungal; Gene Expression Regulation, Fungal - drug effects; GENES; Genes, Fungal; GENETICA; genetics; GENETIQUE; Lac Operon; LIGASAS; LIGASE; messenger RNA; NEUROSPORA CRASSA; Neurospora crassa - enzymology; Neurospora crassa - genetics; Open Reading Frames; Peptides; pharmacology; Phosphotransferases; Phosphotransferases - genetics; polyribosomes; Protein Biosynthesis; Protein Sorting Signals; Protein Sorting Signals - genetics; recombinant DNA; Recombinant Fusion Proteins; Recombinant Fusion Proteins - biosynthesis; reporter genes; RIBOSOMAS; RIBOSOME; structural genes
• ISSN: 0021-9193; 1098-5530; 1067-8832
• DOI: 10.1128/jb.178.8.2172-2177.1996

The Neurospora crassa arg-2 transcript contains an upstream open reading frame (uORF) specifying a 24-residue leader peptide and is subject to a novel form of negative translational regulation in response to arginine. The role of the arg-2 uORF in arginine-specific negative regulation was investigated by using translational fusions of wild-type and mutant arg-2 sequences to the Escherichia coli lacZ reporter gene specifying beta-galactosidase. The wild-type uORF conferred Arg-specific regulation on the reporter gene in N. crassa, but mutated or truncated uORFs did not, as determined by measurements of beta-galactosidase activity produced in N. crassa strains expressing arg-2-lacZ fusion genes. All effects on reporter gene expression were posttranscriptional, as determined by measurement of RNA levels. Both sequence-dependent and sequence-independent effects of uORFs were observed. Genes containing the wild-type uORF or a 21-codon mutated uORF showed reduced translation in comparison with that of a gene lacking a uORF. Both uORF-containing transcripts showed reduced association with polysomes relative to transcripts lacking a uORF, but only the transcript with the wild-type uORF showed a reduced average number of ribosomes associated with it in response to arginine addition. Direct translational fusions between uORF sequences and lacZ sequences indicated that the uORF is translated. Overlapping the uORF with the lacZ initiation codon indicated that ribosome reinitiation at a downstream start codon is not integral to uORF-mediated, Arg-specific translational regulation. These studies provide direct biochemical evidence for arg-2 uORF function in translational control