Characterization of the functional role of nucleotides within the URE2 IRES element and the requirements for eIF2A-mediated repression.
Cap-independent initiation of translation is thought to promote protein synthesis on some mRNAs during times when cap-dependent initiation is down-regulated. However, the mechanism of cap-independent initiation is poorly understood. We have previously reported the secondary structure within the yeast minimal URE2 IRES element. In this study, we sought to investigate the mechanism of internal initiation in yeast by assessing the functional role of nucleotides within the minimal URE2 IRES element, and delineating the cis-sequences that modulate levels of internal initiation using a monocistronic reporter vector. Furthermore, we compared the eIF2A sensitivity of the URE2 IRES element with some of the invasive growth IRES elements using DeltaeIF2A yeast. We found that the stability of the stem-loop structure within the minimal URE2 IRES element is not a critical determinant of optimal IRES activity, and the downstream sequences that modulate URE2 IRES-mediated translation can be defined to discrete regions within the URE2 coding region. Repression of internal initiation on the URE2 minimal IRES element by eIF2A is not dependent on the stability of the secondary structure within the URE2 IRES element. Our data also indicate that eIF2A-mediated repression is not specific to the URE2 IRES element, as both the GIC1 and PAB1 IRES elements are repressed by eIF2A. These data provide valuable insights into the mRNA requirements for internal initiation in yeast, and insights into the mechanism of eIF2A-mediated suppression.
Department of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106, USA.
Lucas C
LC
Reineke
William C
WC
Merrick
D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011061:Poly A / Q000235:genetics; D011328:Prions / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics; D050436:Regulatory Elements, Transcriptional; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D015852:Eukaryotic Initiation Factor-2; D009711:Nucleotides; D011328:Prions; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D011061:Poly A; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae