DNA origami-based single-molecule force spectroscopy elucidates RNA Polymerase III pre-initiation complex stability.
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Date
2020-06-05ICR Author
Author
Kramm, K
Schröder, T
Gouge, J
Vera, AM
Gupta, K
Heiss, FB
Liedl, T
Engel, C
Berger, I
Vannini, A
Tinnefeld, P
Grohmann, D
Type
Journal Article
Metadata
Show full item recordAbstract
The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III.
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Subject
RNA Polymerase III
TATA-Box Binding Protein
Transcription Factor TFIIIB
Recombinant Proteins
DNA, Single-Stranded
Molecular Probes
Microscopy, Confocal
Microscopy, Electron, Transmission
Fluorescence Resonance Energy Transfer
Transcription, Genetic
Nucleic Acid Conformation
Kinetics
Promoter Regions, Genetic
Protein Stability
Single Molecule Imaging
Research team
Vannini Group
Language
eng
Date accepted
2020-05-13
License start date
2020-06-05
Citation
Nature communications, 2020, 11 (1), pp. 2828 - ?
Publisher
NATURE PUBLISHING GROUP