In vivo analysis of Saccharomyces cerevisiae COX2 mRNA 5'-untranslated leader functions in mitochondrial translation initiation and translational activation.
We have used mutational and revertant analysis to study the elements of the 54-nucleotide COX2 5'-untranslated leader involved in translation initiation in yeast mitochondria and in activation by the COX2 translational activator. Pet111p. We generated a collection of mutants with substitutions spanning the entire COX2 5'-UTL by in vitro mutagenesis followed by mitochondrial transformation and gene replacement. The phenotypes of these mutants delimit a 31-nucleotide segment, from -16 to -46, that contains several short sequence elements necessary for COX2 5'-UTL function in translation. The sequences from -16 to -47 were shown to be partially sufficient to promote translation in a foreign context. Analysis of revertants of both the series of linker-scanning alleles and two short deletion/ insertion alleles has refined the positions of several possible functional elements of the COX2 5'-untranslated leader, including a putative RNA stem-loop structure that functionally interacts with Pet111p and an octanucleotide sequence present in all S. cerevisiae mitochondrial mRNA 5'-UTLs that is a potential rRNA binding site.
Dunstan
H M
HM
Section of Genetics and Development, Cornell University, Ithaca, New York 14853-2703, USA.
Green-Willms
N S
NS
Fox
T D
TD
D001483:Base Sequence; D003576:Electron Transport Complex IV / Q000235:genetics*; D005656:Fungal Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D008565:Membrane Proteins; D008928:Mitochondria / Q000502:physiology; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010448:Peptide Initiation Factors; D010641:Phenotype; D014176:Protein Biosynthesis / Q000235:genetics*; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D013489:Suppression, Genetic; D015533:Transcriptional Activation / Q000235:genetics*
D005656:Fungal Proteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; C087048:PET111 protein, S cerevisiae; D010448:Peptide Initiation Factors; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D003576:Electron Transport Complex IV