SEQUENCES-5' OF THE FIRST UPSTREAM OPEN READING FRAME IN GCN4 MESSENGER-RNA 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 transl ate the first of four upstream open reading frames (uORFs) in the mRNA leader resume scanning and can reinitiate downstream, Under non-starv ation conditions reinitiation occurs at one of the remaining three uOR Fs and GCN4 is repressed. Under starvation conditions, in contrast, ri bosomes bypass the uORFs and reinitiate at GCN4 instead, The high freq uency of reinitiation following uORF1 translation depends on an adequa te distance to the next start codon and particular sequences surroundi ng the uORF1 stop codon, We present evidence that sequences 5' to uORF 1 also strongly enhance reinitiation, First, reinitiation was severely inhibited when uORF1 was transplanted into the position of uORF4, eve n though the native sequence environment of the uORF1 stop codon was m aintained, and this effect could not be accounted for by the decreased uORF1-GCN4 spacing, Second, insertions and deletions in the leader pr eceding uORF1 greatly reduced reinitiation at GCN4, Sequences 5' to uO RF1 may influence the probability of ribosome release following peptid e termination at uORF1. Alternatively, they may facilitate rebinding o f an initiation factor required for reinitiation prior to resumption o f the scanning process.