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Antagonistic signals within the COX2 mRNA coding sequence control its translation in Saccharomyces cerevisiae
Elizabeth H Williams1, Thomas D Fox
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853-2703, USA.
Abstract:
Translation of the mitochondrially coded COX2 mRNA within the organelle in yeast produces the precursor of Cox2p (pre-Cox2p), which is processed and assembled into cytochrome c oxidase. The mRNA sequence of the first 14 COX2 codons, specifying the pre-Cox2p leader peptide, was previously shown to contain a positively acting element required for translation of a mitochondrial reporter gene, ARG8(m), fused to the 91st codon of COX2. Here we show that three relatively short sequences within the COX2 mRNA coding sequence, or structures they form in vivo, inhibit translation of the reporter in the absence of the positive element. One negative element was localized within codons 15 to 25 and shown to function at the level of the mRNA sequence, whereas two others are within predicted stem-loop structures formed by codons 22-44 and by codons 46-74. All three of these inhibitory elements are antagonized in a sequence-specific manner by reintroduction of the upstream positive-acting sequence. These interactions appear to be independent of 5'- and 3'-untranslated leader sequences, as they are also observed when the same reporter constructs are expressed from the COX3 locus. Overexpression of MRS2, which encodes a mitochondrial magnesium carrier, partially suppresses translational inhibition by each isolated negatively acting element, but does not suppress them in combination. We hypothesize that interplay among these signals during translation in vivo may ensure proper timing of pre-Cox2p synthesis and assembly into cytochrome c oxidase.
Insights
Mitochondrial COX2 mRNA translation in yeast is regulated by both positive and negative elements. These elements control the synthesis of cytochrome c oxidase precursor protein (pre-Cox2p) and its assembly.
Area of Science:
- Mitochondrial Gene Expression
- Protein Synthesis Regulation
- Yeast Molecular Biology
Background:
- Mitochondrial translation of COX2 mRNA in yeast produces the precursor of Cox2p (pre-Cox2p).
- A previously identified positive element in the COX2 mRNA leader sequence is crucial for reporter gene translation.
- Cytochrome c oxidase is essential for cellular respiration.
Purpose of the Study:
- To identify and characterize negative regulatory elements within the COX2 mRNA coding sequence that inhibit translation.
- To investigate the interplay between positive and negative regulatory elements in COX2 mRNA translation.
- To explore the role of MRS2 in modulating mitochondrial translation.
Main Methods:
- Construction and expression of reporter gene fusions with COX2 mRNA sequences in yeast.
- Localization of negative elements within specific COX2 mRNA codons and predicted stem-loop structures.
- Analysis of translational inhibition and its antagonism by reintroducing the positive element.
- Assessment of MRS2 overexpression effects on translational inhibition.
Main Results:
- Three distinct negative regulatory elements within the COX2 mRNA coding sequence were identified, inhibiting reporter translation.
- One element functions at the mRNA sequence level (codons 15-25), while two others reside in predicted stem-loop structures (codons 22-44 and 46-74).
- These inhibitory elements are antagonized by the upstream positive-acting sequence in a sequence-specific manner.
- MRS2 overexpression partially suppresses individual negative elements but not their combined effect.
Conclusions:
- The COX2 mRNA coding sequence contains inhibitory elements that regulate pre-Cox2p synthesis.
- Interplay between positive and negative regulatory signals likely ensures precise timing of pre-Cox2p synthesis and cytochrome c oxidase assembly.
- Mitochondrial magnesium carrier MRS2 may play a role in modulating translational control.