Determinants for binding of a 40 kDa protein to the leaders of yeast mitochondrial mRNAs.
An abundant yeast mitochondrial 40 kDa protein (p40) binds with high specificity to the 5'-untranslated region of cytochrome c oxidase subunit II (COX2) mRNA. Using mobility shift and competition assays, we show here that purified p40 complexes with the leaders of all eight mitochondrial mRNAs of Saccharomyces cerevisiae. The location of the protein binding site on the different leaders is not conserved with respect to the AUG start codon. In vitro RNA footprint and deletion experiments have been used to define the p40-binding site on the leaders of COX1 and ATP9 mRNAs. Nucleotides at, and near, a single stranded region are protected or exposed for DEPC modification by binding of p40 to these leaders. Removal of this region from the COX1 messenger shows that it is essential for the protein-RNA interaction. While no obvious sequence similarity can be detected between the single stranded regions in different leaders, a nearby helical segment is conserved. A consensus model for p40-RNA interactions is presented and the possible biological function of p40 is discussed.
Dekker
P J
PJ
Department of Molecular Cell Biology, University of Amsterdam, The Netherlands.
Stuurman
J
J
van Oosterum
K
K
Grivell
L A
LA
D001483:Base Sequence; D001665:Binding Sites; D004272:DNA, Mitochondrial / Q000235:genetics*; D005656:Fungal Proteins / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship
D004272:DNA, Mitochondrial; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D012331:RNA, Fungal; D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D016601:RNA-Binding Proteins; D012313:RNA