EFFECT OF SEQUENCE CONTEXT AT STOP CODONS ON EFFICIENCY OF REINITIATION IN GCN4 TRANSLATIONAL CONTROL

Translational control of the GCN4 gene involves two short open reading frames in the mRNA leader (uORF1 and uORF4) that differ greatly in th e ability to allow reinitiation at GCN4 following their own translatio n. The low efficiency of reinitiation characteristic of uORF4 can be r econstituted in a hybrid element in which the last codon of uORF1 and 10 nucleotides 3' to its stop codon (the termination region) are subst ituted with the corresponding nucleotides from uORF4. To define the fe atures of these 13 nucleotides that determine their effects on reiniti ation, we separately randomized the sequence of the third codon and te rmination region of the uORF1-uORF4 hybrid and selected mutant alleles with the high-level reinitiation that is characteristic of uORF1. The results indicate that many different A+U-rich triplets present at the third codon of uORF1 can overcome the inhibitory effect of the termin ation region derived from uOPF4 on the efficiency of reinitiation at G CN4. Efficient reinitiation is not associated with codons specifying a particular amino acid or isoacceptor tRNA. Similarly, we found that a diverse collection of A+U-rich sequences present in the termination r egion of uORF1 could restore efficient reinitiation at GCN4 in the pre sence of the third codon derived from uORF4. To explain these results, we propose that reinitiation can be impaired by stable base pairing b etween nucleotides flanking the uORF1 stop codon and either the tRNA w hich pairs with the third codon, the rRNA, or sequences located elsewh ere in GCN4 mRNA. We suggest that these interactions delay the resumpt ion of scanning following peptide chain termination at the uORF and th ereby lead to ribosome dissociation from the mRNA.