The secondary structure and sequences influencing the expression and selection of the AUG initiator codon in the yeast Saccharomyces cerevisiae were investigated with two fused genes, which were composed of either the CYC7 or CYC1 leader regions, respectively, linked to the lacZ coding region. In addition, the strains contained the upf1-delta disruption, which stabilized mRNAs that had premature termination codons, resulting in wild-type levels. The following major conclusions were reached by measuring beta-galactosidase activities in yeast strains having integrated single copies of the fused genes with various alterations in the 89 and 38 nucleotide-long untranslated CYC7 and CYC1 leader regions, respectively. The leader region adjacent to the AUG initiator codon was dispensable, but the nucleotide preceding the AUG initiator at position -3 modified the efficiency of translation by less than twofold, exhibiting an order of preference A > G > C > U. Upstream out-of-frame AUG triplets diminished initiation at the normal site, from essentially complete inhibition to approximately 50% inhibition, depending on the position of the upstream AUG triplet and on the context (-3 position nucleotides) of the two AUG triplets. In this regard, complete inhibition occurred when the upstream and downstream AUG triplets were closer together, and when the upstream and downstream AUG triplets had, respectively, optimal and suboptimal contexts. Thus, leaky scanning occurs in yeast, similar to its occurrence in higher eukaryotes. In contrast, termination codons between two AUG triplets causes reinitiation at the downstream AUG in higher eukaryotes, but not generally in yeast. Our results and the results of others with GCN4 mRNA and its derivatives indicate that reinitiation is not a general phenomenon in yeast, and that special sequences are required.

The cycl-512 mutation is a 38-bp deletion in the 3' untranslated region of the CYCI gene, which encodes iso-l-cytochrome c in Saccharomyces cerevisiae. This deletion caused a 90% reduction in the levels of the CYCI mRNA and protein because of the absence of the normal 3' end-forming signal. Although the 3' end-forming signal was not defined by previous The proper formation of mRNA 3' ends and transcription termination are essential for the optimal expression of genes in eukaryotes. Point mutations in the mRNA 3' end-forming signal, or a -deletion of the signal, can lead to drastic decreases in gene expression (1-4). Such a case was found in the yeast cycl-512 mutant (5). In addition to displaying a 90% decrease in CYC1+ mRNA and iso-1-cytochrome c, the cycl-512 mutation also caused the formation of aberrantly long transcripts with many discrete 3' termini ranging from the wild-type poly(A) site to endpoints >2000 nt downstream (5-9). Butler and Platt (10) found that the same 38-bp region deleted in cycl-512 was necessary for the proper cleavage of extended CYCI transcripts in vitro. In addition, the same 38-bp region was found by Russo and Sherman (11) to be able to cause the termination of transcription in vivo. Although this cycl-512 mutation and its properties were uncovered >10 years ago (5), and although this cycl-512 mutation was instrumental for defining 3' end-forming signals by examining sequences that restored the levels of the CYCI mRNAs (6), the exact 3' end-forming signals of the wild-type CYC1 gene were not previously determined. Because of the inability to inactivate the 3' end-forming signal by nested deletions and multiple base-pair substitution within the 38-bp region, workers suggested that the 3' end-forming signal was only an A+T-rich region (7) or a symmetrical RNA structure (8). Russo et al (9) suggested that the CYC1 3' end-forming signals may have functional redundancy, whereas Wu (12) suggested three TA pairs may be important.Signals required for 3' end formation were uncovered by examining intragenic revertants and oligonucleotide-directed alterations that restored the cycl-512 defect (6,9

The steady-state levels and half-lives of CYC1 mRNAs were estimated in a series of mutant strains of Saccharomyces cerevisiae containing (i) TAA nonsense codons, (ii) ATG initiator codons, or (iii) the sequence ATA ATG ACT TAA (denoted ATG-TAA) at various positions along the CYC1 gene, which encodes iso-1-cytochrome c. These mutational alterations were made in backgrounds lacking all internal in-frame and out-of-frame ATG triplets or containing only one ATG initiator codon at the normal position. The results revealed a ''sensitive'' region encompassing approximately the first half of the CYC1 mRNA, in which nonsense codons caused Upf1-dependent degradation. This result and the stability of CYC1 mRNAs lacking all ATG triplets, as well as other results, suggested that degradation occurs unless elements associated with this sensitive region are covered with 80S ribosomes, 40S ribosomal subunits, or ribonucleoprotein particle proteins. While elongation by 80S ribosomes could be prematurely terminated by TAA codons, the scanning of 40S ribosomal units could not be terminated solely by TAA codons but could be disrupted by the ATG-TAA sequence, which caused the formation and subsequent prompt release of 80S ribosomes. The ATG-TAA sequence caused degradation of the CYC1 mRNA only when it was in the region spanning nucleotide positions ؊24 to ؉37 but not in the remaining 3 distal region, suggesting that translation could initiate only in this restricted initiation region. CYC1 mRNA distribution on polyribosomes confirmed that only ATG codons within the initiation region were translated at high efficiency. This initiation region was not entirely dependent on the distance from the 5 cap site and was not obviously dependent on the short-range secondary structure but may simply reflect an open structural requirement for initiation of translation of the CYC1 mRNA.Mutationally-altered forms of the CYC1 gene, which encodes iso-1-cytochrome c in the yeast Saccharomyces cerevisiae have been used to elucidate the major features of translation. The deficiencies of iso-1-cytochrome c in certain cyc1 mutants and the corresponding deduced DNA sequences of cyc1 mutations indicated that AUG was the only codon capable of initiating translation at appreciable levels (48, 49), a finding confirmed in yeast cells with other systems (7, 9). Also, early studies indicated that translation of CYC1 mRNA initiated at the most upstream AUG codon (45-47) and that translation could initiate at a relocated AUG initiator codon within a 37-nucleotide region around the normal initiation site (45-49). These results and the results with deletion mutants (2) suggested the lack of any additional sequence requirements for initiation of translation. However, nucleotides preceding the AUG initiator codon at positions Ϫ1 and Ϫ3 slightly influenced initiation of translation, and the 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 hairpi...

Several studies have indicated that degradation of certain mRNAs is tightly coupled to their translation, whereas, in contrast, other observations suggested that translation can be inhibited without changing the stability of the mRNA. We have addressed this question with the use of altered CYCI alleles, which encode iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. The cycl-1249 mRNA, which lacks all in-frame and out-of-frame AUG triplets, was as stable as the normal mRNA. This finding established that translation is not required for the degradation of CYCI mRNAs. Furthermore, poly(G)18 tracks were introduced within the CYCI mRNA translated regions to block exonuclease degradation. The recovery of 3' fragments revealed that the translatable and the AUG-deficient mRNAs are both degraded 5' -> 3'. Also, the increased stability of CYCI mRNAs in xrnl-A strains lacking Xrnlp, the major 5' --3' exonuclease, established that the normal and AUG-deficient mRNAs are degraded by the same pathway. In addition, deadenylylation, which activates the action of Xrnlp, occurred at equivalent rates in both normal and AUG-deficient mRNAs. We conclude that translation is not required for the normal degradation of CYCI mRNAs, and that translatable and untranslated mRNAs are degraded by the same pathway. mRNA turnover is important for determining the levels and regulation of gene expression. In general, the rate at which a particular protein is made is proportional to the cytoplasmic level of the corresponding mRNA. Thus, the use of mRNA requires the proper coordination of transcription, translation, and mRNA turnover. The degradation of mRNA in eukaryotic cells is initiated by endonucleolytic cleavage (1, 2) or by shortening of the poly(A) tails (3-6) that, for some mRNAs, activates a deadenylylation-dependent decapping reaction (7) and subsequently is followed by 5' -> 3' exonucleolytic deg-