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High-throughput Purification of Affinity-tagged Recombinant Proteins
Published on: August 26, 2012
Structural characterization of human general transcription factor TFIIF in solution
Satoko Akashi1, Shinjiro Nagakura, Seiji Yamamoto
1International Graduate School of Arts and Sciences, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan.
Protein Science : a Publication of the Protein Society
|January 26, 2008
Summary
Human general transcription factor IIF (TFIIF) is an alphabeta heterodimer, confirmed by mass spectrometry. This protein complex plays a role in RNA polymerase II transcription initiation but shows no direct interaction with TFIIE in solution.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- General transcription factor IIF (TFIIF) is crucial for RNA polymerase II (Pol II) transcription initiation.
- TFIIF is a component of the transcription pre-initiation complex (PIC).
- Previous studies suggested TFIIF's size was over 200 kDa based on SEC.
Purpose of the Study:
- To characterize the subunit composition and molecular size of human TFIIF.
- To investigate the interaction between human TFIIF and human TFIIE.
- To clarify TFIIF's role in the transcription pre-initiation complex.
Main Methods:
- Recombinant expression and purification of human TFIIF.
- Size-exclusion chromatography (SEC).
- Electrospray ionization mass spectrometry (ESI-MS).
- Chemical cross-linking and MALDI-MS.
Main Results:
- Purified recombinant TFIIF exhibited similar transcription activity to natural TFIIF.
- ESI-MS revealed a molecular size of 87 kDa, indicating an alphabeta heterodimer.
- Cross-linking and MALDI-MS confirmed the alphabeta heterodimer structure.
- No direct interaction was detected between human TFIIF and TFIIE in solution.
Conclusions:
- Human TFIIF exists as an alphabeta heterodimer, contrary to previous size estimations.
- This heterodimeric structure is likely maintained within the transcription pre-initiation complex.
- TFIIF and TFIIE do not directly interact in solution and may associate with DNA separately.
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