Initiation of translation can occur only in a restricted region of the CYC1 mRNA of Saccharomyces cerevisiae.
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 -27 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.
Yun
D F
DF
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642.
Sherman
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism; D003062:Codon / Q000235:genetics; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000235:genetics; D045304:Cytochromes c; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010442:Peptide Chain Initiation, Translational; D017354:Point Mutation; D011132:Polyribosomes / Q000378:metabolism* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013045:Species Specificity
C014059:CYC1 protein, S cerevisiae; D003062:Codon; D003574:Cytochrome c Group; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c