The mRNA sequence and structures that modify and are required for translation of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae were investigated with sets of CYCI alleles having alterations in the 5' leader region. Measurements of levels of CYCI mRNA and iso-1-cytochrome c in strains having single copies of altered alleles with nested deletions led to the conclusion that there is no specific sequence adjacent to the AUG initiator codon required for efficient translation. However, the nucleotides preceding the AUG initiator codon at positions -1 and -3 slightly modified the efficiency of translation to an order of preference similar to that found in higher cells. In contrast to large effects observed in higher eucaryotes, the magnitude of this AUG context effect in S. cerevisiae was only two-to threefold. Furthermore, introduction of hairpin structures in the vicinity of the AUG initiator codon inhibited translation, with the degree of inhibition related to the stability and proximity of the hairpin. These results with S. cerevisiae and published findings on other organisms suggest that translation in S. cerevisiae is more sensitive to secondary structures than is translation in higher eucaryotes.Mutational alterations at the end of the CYCI gene in Saccharomyces cerevisiae previously have been used to investigate the nucleotide sequences in mRNA that are required for translation of its gene product, iso-1-cytochrome c. Protein analysis of iso-1-cytochrome c from certain cycl revertants and the corresponding deduced DNA sequences of the cycl mutations led Stewart et al. (54) and Sherman and Stewart (45) to conclude that AUG was the only codon capable of initiating translation at appreciable levels. Of the remaining 63 codons, only the UGG codon was not excluded as being a potential initiator codon (41). Early work also indicated that translation of CYC1 mRNA initiated at the most 5'-proximal AUG (43-45). Furthermore, translation could initiate with equal or nearly equal efficiency at a relocated AUG initiator codon within a 37-nucleotide region around the normal initiation site (43)(44)(45)54), suggesting a lack of any additional sequence requirements for initiation in S. cerevisiae analogous to the essential ribosome-binding site present in bacterial mRNA (53). However, the nucleotides immediately 5' to the AUG initiator codon in CYCI mRNA were observed to influence the efficiency of translation slightly, although precise rules were not established (45).In the present study, we analyzed the structural elements that influence the translational efficiency of CYCI mRNA. Earlier studies relied on mutation and recombination produced in vivo, whereas here specific alterations within the 5'-noncoding region of CYCI were produced primarily in vitro. The levels of CYCI mRNA and iso-1-cytochrome c were determined in isogenic strains, each carrying a single copy of an altered CYCI gene at the normal chromosomal position. Our results demonstrated a lack of any specific sequence requirement in the 5'-untranslated ...