Identification and characterization of upstream open reading frames (uORF) in the 5' untranslated regions (UTR) of genes in Saccharomyces cerevisiae.
We have taken advantage of recently sequenced hemiascomycete fungal genomes to computationally identify additional genes potentially regulated by upstream open reading frames (uORFs). Our approach is based on the observation that the structure, including the uORFs, of the post-transcriptionally uORF regulated Saccharomyces cerevisiae genes GCN4 and CPA1 is conserved in related species. Thirty-eight candidate genes for which uORFs were found in multiple species were identified and tested. We determined by 5' RACE that 15 of these 38 genes are transcribed. Most of these 15 genes have only a single uORF in their 5' UTR, and the length of these uORFs range from 3 to 24 codons. We cloned seven full-length UTR sequences into a luciferase (LUC) reporter system. Luciferase activity and mRNA level were compared between the wild-type UTR construct and a construct where the uORF start codon was mutated. The translational efficiency index (TEI) of each construct was calculated to test the possible regulatory function on translational level. We hypothesize that uORFs in the UTR of RPC11, TPK1, FOL1, WSC3, and MKK1 may have translational regulatory roles while uORFs in the 5' UTR of ECM7 and IMD4 have little effect on translation under the conditions tested.
Zhang
Zhihong
Z
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Dietrich
Fred S
FS
D020121:5' Untranslated Regions / Q000235:genetics*; D050976:Basic-Leucine Zipper Transcription Factors; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics; D030561:Databases, Nucleic Acid; D020869:Gene Expression Profiling; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics*; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics
D020121:5' Untranslated Regions; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C403521:CPA1 protein, S cerevisiae; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)