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The polypyrimidine tract binding protein is a monomer
Tom P Monie1, Helena Hernandez, Carol V Robinson
1Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, United Kingdom.
RNA (New York, N.Y.)
|November 30, 2005
Summary
Polypyrimidine tract binding (PTB) protein, crucial for mRNA splicing, is monomeric in cells. Previous studies misidentified it as a dimer due to oxidizing conditions causing disulfide bonds.
Area of Science:
- Molecular Biology
- Protein Biochemistry
Background:
- Polypyrimidine tract binding (PTB) protein regulates alternative mRNA splicing, translation initiation, and polyadenylation.
- Previous research suggested PTB exists as a dimer, influencing mechanistic models of its function.
Purpose of the Study:
- To investigate the oligomeric state of full-length PTB protein.
- To provide biophysical and biochemical evidence clarifying PTB's molecular status in solution.
Main Methods:
- Blue-native polyacrylamide gel electrophoresis (BN-PAGE)
- Size-exclusion chromatography (SEC)
- SDS-PAGE
- Time-of-flight electrospray ionization mass spectrometry (TOF-ESI-MS)
Main Results:
- PTB exists as a single molecular species under native reducing conditions.
- Oxidizing conditions revealed a larger species, identified as a disulfide-linked PTB dimer mediated by Cys23.
- SDS-PAGE and mass spectrometry confirmed the reduced species as monomeric PTB.
Conclusions:
- The monomeric state of PTB is supported by new biophysical and biochemical evidence.
- Previous misidentification of PTB as a dimer likely resulted from the use of oxidizing environments.
- PTB functions as a monomer in the reducing intracellular environment, impacting models of mRNA metabolism regulation.