The vectors were constructed using recombinant DNA fragments generated via PCR using oligodeoxyribonucleotide primers specific for YAP sequences as well as synthetic DNA as building blocks. The plasmids have been named according to a systematic nomenclature described previously (Vilela et al., 1998). All sequences were inserted into YCpSUPEX1 (GPF promoter; Oliveira et al., 1993b) and/or YCp22FL (TEF1 promoter; Oliveira et al., 1993a) and verified by means of DNA sequencing. Six genes were used: the genes encoding firefly luciferase (LUC) and bacterial chloramphenicol acetyl transferase (cat), and S.cerevisiae YAP1 and YAP2, GCN4 and the modified version of the PGK1 gene, pRIPPGKH2(3)Δ1. The latter gene was kindly donated by Dr Alan Jacobson (Peltz et al., 1993a). The yeast genes were inserted into the YCp22FL vector after introduction of the NdeI and XbaI sites at the 5′ and 3′ ends of the genes' main ORFs. Constructs pY1, pY2, pΔuY1, pΔu1Y2, pΔu2Y2, pΔu(1+2)Y2, puY1du4G4, pmuY1Δdu4G4, pAuY1du4G4, pAmuY1du4G4, pu4G4 were described previously (Vilela et al., 1998; Table I). pΔAmuY1Δdu4G4 was used as a control for pAmuY1Δdu4G4 and was constructed from pAmuY1Δdu4G4 by mutation of the uORF start codon by a single base change (AUGAAG). puY1u1Y2 resulted from the insertion at position +112 nt in puY1 of the YAP2 uORF1 and the 38 nt downstream of this element, resulting in a construct containing both the YAP1 uORF and YAP2 uORF1. By means of a single base deletion, the YAP2 uORF was placed out of frame relative to the YAP1 uORF1 creating construct puY1fu1Y2. pΔuY1u1Y2 was generated by mutation of the YAP1 uORF start codon (AUGAAG) in puY1u1Y2. As a control, the YAP2 uORF start codon was subsequently mutated (AUGAAG), generating pΔuY1Δu1Y2. In a further control construct, the nucleotide context of the YAP1 uORF was changed, generating pAmuY1du4G4u1Y2 (pAmuY1du4G4 is described above; Vilela et al., 1998). Constructs puY160u1Y2 and puY1u1Y260 were derived from puY1u1Y2 by inserting, respectively, a 60 nucleotide spacer (Table II) between the restriction sites XhoI site and NdeI. Similarly, puY160u1Y260 resulted from the insertion of both spacers at the XhoI and NdeI sites of puY1u1Y2 (Figure 5A). pGCN4 contains the wild-type GCN4 leader in which the start codons of uORF2 and uORF3 were mutated (Abastado et al., 1991). The vectors described in Figure 3 refer to the combination of different synthetic fragments introduced into YCpSUPEX: the first module, cloned between the BamHI and XhoI sites, contains an uORF (YAP1 uORF, YAP2 uORF1, GCN4 uORF1 or GCN4 uORF4) as well as the 10 nucleotides downstream of each uORF, creating constructs puY1, pu1Y2, pu1G4 and pu4G4. The various uORFs were cloned between the restriction sites BglII and XhoI. The second module was cloned between the restriction sites XhoI and NdeI and contains either an unstructured leader (pcat; Figure 3A) or a stem–loop structure with a predicted stability of −28.8 kcal/mol, 5 nucleotides upstream of the cat start codon (pScat; Figure 3B). pΔu4G4. cat results from mutation of the GCN4 uORF4 start codon in pu4G4. cat (AUGAAG) (Figure 3B). Constructs puY160Scat and puY130Scat were made from puY1. cat by inserting, respectively, a 60 nucleotide spacer in the XhoI site (Figure 4). As a control, the −28.8 kcal/mol stem–loop was replaced by a stem–loop of a stability of 8.6 kcal/mol, generating construct puY2. cat (Figure 4A). All of the leader sequences based on synthetic DNA fragments are listed in Table II. The constructs depicted in Figure 7 represent combinations of the leaders pY1, pY2, pu4G4, pΔu2Y2 and pΔu(1+2)Y2 (described above) with the GCN4 reading frame. The pS leader contains a stem–loop with a predicted stability of −25.5 kcal/mol introduced upstream of the GCN4 ORF. Finally, construct pu4S refers to the GCN4 mRNA containing only uORF4 combined with a stem–loop structure with a predicted stability of −8.7 kcal/mol. This plasmid was generated using constructs pA50 and p237, kindly given to us by Dr Alan Hinnebusch (Abastado et al., 1991).