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DNA-binding properties and characterization of human transcription factor TFIIIC2
P A Boulanger1, S K Yoshinaga, A J Berk
1Molecular Biology Institute, University of California, Los Angeles 90024.
The Journal of Biological Chemistry
|November 5, 1987
Summary
Human RNA polymerase III transcription factor C2 (TFIIIC2) specifically binds adenovirus VA1 DNA's B-block region with high affinity. This interaction is crucial for regulating viral gene transcription by TFIIIC2, a protein complex potentially dimerized from 250-kDa subunits.
Area of Science:
- Molecular Biology
- Transcription Factor Analysis
- Adenovirus Gene Regulation
Background:
- Adenovirus VA1 DNA contains a regulatory B-block region essential for transcription.
- Human transcription factor IIIC2 (TFIIIC2) is involved in RNA polymerase III-mediated transcription.
- Understanding the interaction between TFIIIC2 and VA1 DNA is key to deciphering viral gene expression.
Purpose of the Study:
- To investigate the specific binding interaction between TFIIIC2 and the B-block region of adenovirus VA1 DNA.
- To characterize the binding affinity and stoichiometry of the TFIIIC2-VA1 DNA complex.
- To determine the molecular properties and subunit composition of TFIIIC2.
Main Methods:
- Gel DNA-binding assays, including DNase I footprinting and gel shift assays.
- Competition experiments using mutated and truncated VA1 DNA probes.
- Equilibrium binding constant determination, sucrose gradient sedimentation, and UV cross-linking followed by SDS-PAGE.
Main Results:
- TFIIIC2 binds the VA1 B-block region with high affinity (Ks = 2 x 10(11) M-1) compared to non-specific DNA (Kn = 6 x 10(4) M-1).
- Approximately 1500 TFIIIC2 molecules were extracted per 293 cell, with binding sites [C0] ranging from 0.6 to 1.6 x 10(-10) M.
- Sedimentation and UV cross-linking suggest TFIIIC2 is a dimer of a ~250-kDa polypeptide, with a native molecular weight of 400-500 kDa.
Conclusions:
- TFIIIC2 exhibits high-affinity, specific binding to the adenovirus VA1 DNA B-block, indicating a critical role in viral transcription initiation.
- The study quantifies the binding parameters and estimates the cellular abundance of TFIIIC2.
- Evidence supports TFIIIC2 being a homodimer composed of approximately 250-kDa subunits.