Purification and characterization of an RNA dodecamer sequence binding protein from mitochondria of Saccharomyces cerevisiae
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Updated: Oct 19, 2026
Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation
Published on: March 14, 2014
1Department of Molecular Microbiology and Immunology, St. Louis University Health Sciences Center, St. Louis, Missouri, 63104, USA.
Abstract:
Saccharomyces cerevisiae mitochondrial mRNAs terminate at their 3' ends with a conserved dodecamer sequence, 5'-AAUAA(U/C)AUUCUU-3'. We have identified a nuclear-encoded protein (DBP) which specifically binds to the dodecamer sequence and have purified it to apparent homogeneity by RNA affinity chromatography. DBP consists of a single polypeptide of 55 kDa and binds to its RNA substrate with a 1:1 stoichiometry. Scatchard analysis determines that K(d) is 0.93 nM for the canonical dodecamer sequence (5'-AAUAAUAUUCUU-3') and 0.46 nM for the only naturally occurring variant (5'-AAUAACAUUCUU-3') unique to oli1 gene. Based on the studies using mutant oligonucleotides, DBP appears to recognize primarily the nucleotide sequence of an RNA rather than its potential secondary structure.
Insights
Researchers identified a nuclear-encoded protein (DBP) that binds to a specific sequence in Saccharomyces cerevisiae mitochondrial messenger RNA (mRNA) 3' ends. This protein, DBP, recognizes the RNA sequence, not its structure, aiding in mRNA termination.
Area of Science:
- Molecular Biology
- Yeast Genetics
- RNA Biology
Background:
- Mitochondrial messenger RNAs (mRNAs) in Saccharomyces cerevisiae exhibit a conserved 3' terminal dodecamer sequence (5'-AAUAA(U/C)AUUCUU-3').
- Understanding the factors involved in mRNA 3' end processing is crucial for gene expression regulation in mitochondria.
Purpose of the Study:
- To identify and characterize nuclear-encoded proteins that specifically bind to the conserved dodecamer sequence in yeast mitochondrial mRNAs.
- To elucidate the binding characteristics and sequence specificity of the identified protein.
Main Methods:
- RNA affinity chromatography was employed to purify the protein that binds to the dodecamer sequence.
- Biochemical assays, including Scatchard analysis, were used to determine binding affinity and stoichiometry.
- Studies utilizing mutant oligonucleotides were performed to assess sequence versus structure recognition.
Main Results:
- A nuclear-encoded protein, designated DBP, was identified and purified.
- DBP is a 55 kDa polypeptide that binds the dodecamer RNA sequence with high affinity (K(d) values in the nanomolar range).
- Binding affinity is higher for the canonical dodecamer and a naturally occurring variant (oli1 gene) compared to other sequences, indicating sequence-specific recognition.
Conclusions:
- DBP is a specific DNA/RNA binding protein involved in the 3' end processing of Saccharomyces cerevisiae mitochondrial mRNAs.
- DBP recognizes the nucleotide sequence of the RNA rather than its secondary structure.
- This finding contributes to the understanding of post-transcriptional regulation in mitochondria.
