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A post-recruitment function for the RNA polymerase III transcription-initiation factor IIIB
G A Kassavetis1, A Kumar, G A Letts
1Department of Biology and Center for Molecular Genetics, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA.
Summary
Mutant transcription factor (TF) IIIB components Brf and B" are active on supercoiled DNA but not linear DNA. This suggests TFIIIB actively opens DNA during transcription initiation by RNA polymerase III.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Transcription factor (TF) IIIB is essential for RNA polymerase (pol) III-dependent transcription.
- TFIIIB comprises TATA-binding protein, TFIIB-related factor (Brf), and pol III-specific subunit B".
Purpose of the Study:
- To investigate the role of specific TFIIIB mutations in Saccharomyces cerevisiae transcription.
- To elucidate the mechanism of TFIIIB in promoter opening during transcription initiation.
Main Methods:
- Site-directed mutagenesis of Brf and B" subunits in yeast.
- In vitro transcription assays using supercoiled and linear DNA templates.
- Analysis of TFIIIB-DNA-pol III complex formation and promoter opening.
Main Results:
- Mutant TFIIIB proteins retained activity on supercoiled DNA but were inactive on linear DNA.
- Inactive complexes successfully recruited and positioned pol III at the transcription start site.
- Mutant complexes failed to form DNA strand-separated open promoter complexes.
Conclusions:
- TFIIIB plays an active role in promoter opening, a critical step in transcription initiation.
- The DNA topology (supercoiled vs. linear) is crucial for TFIIIB function in promoter opening.
- Understanding TFIIIB's mechanism provides insights into RNA polymerase III transcription regulation.