Sequences 5' of the first upstream open reading frame in GCN4 mRNA are required for efficient translational reinitiation.
Translation of yeast GCN4 mRNA occurs by a reinitiation mechanism that is modulated by amino acid levels in the cell. Ribosomes which translate the first of four upstream open reading frames (uORFs) in the mRNA leader resume scanning and can reinitiate downstream. Under non-starvation conditions reinitiation occurs at one of the remaining three uORFs and GCN4 is repressed. Under starvation conditions, in contrast, ribosomes bypass the uORFs and reinitiate at GCN4 instead. The high frequency of reinitiation following uORF1 translation depends on an adequate distance to the next start codon and particular sequences surrounding the uORF1 stop codon. We present evidence that sequences 5' to uORF1 also strongly enhance reinitiation. First, reinitiation was severely inhibited when uORF1 was transplanted into the position of uORF4, even though the native sequence environment of the uORF1 stop codon was maintained, and this effect could not be accounted for by the decreased uORF1-GCN4 spacing. Second, insertions and deletions in the leader preceding uORF1 greatly reduced reinitiation at GCN4. Sequences 5' to uORF1 may influence the probability of ribosome release following peptide termination at uORF1. Alternatively, they may facilitate rebinding of an initiation factor required for reinitiation prior to resumption of the scanning process.
D000483:Alleles; D003062:Codon; D004268:DNA-Binding Proteins; D015247:Deoxyribonuclease HindIII; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D017353:Gene Deletion; D015870:Gene Expression; D018014:Gene Transfer Techniques; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics; D015347:RNA Probes; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D003062:Codon; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D021382:Protein Sorting Signals; D015347:RNA Probes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; D015247:Deoxyribonuclease HindIII