title: An upstream ORF with non-AUG start codon is translated in vivo but dispensable for translational control of GCN4 mRNA.
abstract: Genome-wide analysis of ribosome locations in mRNAs of Saccharomyces cerevisiae has revealed the translation of upstream open reading frames that initiate with near-cognate start codons in many transcripts. Two such non-translation initiation codon (AUG)-initiated upstream open reading frames (uORFs) (nAuORFs 1 and 2) occur in GCN4 mRNA upstream of the four AUG-initiated uORFs (uORFs 1-4) that regulate GCN4 translation. We verified that nAuORF2 is translated in vivo by demonstrating β-galactosidase production from lacZ coding sequences fused to nAuORF2, in a manner abolished by replacing its non-AUG initiation codon (AUA) start codon with the non-cognate triplet AAA, whereas translation of nAuORF1 was not detected. Importantly, replacing the near-cognate start codons of both nAuORFs with non-cognate triplets had little or no effect on the repression of GCN4 translation in non-starved cells, nor on its derepression in response to histidine limitation, nutritional shift-down or treatment with rapamycin, hydrogen peroxide or methyl methanesulfonate. Additionally, we found no evidence that initiation from the AUA codon of nAuORF2 is substantially elevated, or dependent on Gcn2, the sole eIF2α kinase of yeast, in histidine-deprived cells. Thus, although nAuORF2 is translated in vivo, it appears that this event is not stimulated by eIF2α phosphorylation nor significantly influences GCN4 translational induction under various starvation or stress conditions.
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice K Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Alan G
authors.initials: AG
authors.lastname: Hinnebusch
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics; D000619:Aminohydrolases / Q000235:genetics; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D018387:Codon, Initiator; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D011755:Pyrophosphatases / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D018387:Codon, Initiator; C501391:GCN4 protein, S cerevisiae; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D000429:Alcohol Oxidoreductases; C092116:HIS4 protein, S cerevisiae; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D000619:Aminohydrolases; D011755:Pyrophosphatases