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High-throughput Purification of Affinity-tagged Recombinant Proteins
Published on: August 26, 2012
MCM proteins are associated with RNA polymerase II holoenzyme.
K Yankulov1, I Todorov, P Romanowski
1Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario N1G 2W1, Canada. yankulov@uoguelph.ca
Molecular and Cellular Biology
|August 24, 1999
Summary
Minichromosome maintenance proteins (MCMs) are newly found to be involved in RNA polymerase II (Pol II) transcription. These DNA replication proteins associate with the Pol II holoenzyme complex, suggesting a novel role in gene expression.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Minichromosome maintenance proteins (MCMs) are essential for DNA replication initiation.
- MCM proteins function as ATP-dependent helicases and interact with origin recognition complexes.
Purpose of the Study:
- To investigate a potential role for MCM proteins beyond DNA replication.
- To determine if MCM proteins interact with the RNA polymerase II (Pol II) transcriptional machinery.
Main Methods:
- Microinjection of antibodies against MCM2 into Xenopus oocytes.
- Co-purification of MCM proteins with Pol II and general transcription factors (GTFs).
- Immunoaffinity chromatography using anti-MCM3 antibodies and C-terminal domain (CTD) affinity columns.
Main Results:
- Antibodies against MCM2 inhibited Pol II transcription in Xenopus oocytes.
- MCM proteins were found to co-purify with Pol II and GTFs in high-molecular-weight holoenzyme complexes.
- MCMs associated with the Pol II holoenzyme complex, and this interaction depended on the CTD of Pol II.
Conclusions:
- MCM proteins are components of the Pol II transcriptional apparatus.
- MCMs have a novel function in regulating gene transcription.
- The association of MCMs with the holoenzyme complex is mediated by the CTD of Pol II.
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