title: Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.
abstract: Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A "step-wise" preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB-promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II-TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.
authors: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA.
authors: Zhengjian
authors: Z
authors: Zhang
authors: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA.
authors: Brian P
authors: BP
authors: English
authors: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA.
authors: Jonathan B
authors: JB
authors: Grimm
authors: Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037 USA.
authors: Stephanie A
authors: SA
authors: Kazane
authors: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA.
authors: Wenxin
authors: W
authors: Hu
authors: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA.
authors: Albert
authors: A
authors: Tsai
authors: Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA.|Li Ka Shing Center for Biomedical and Health Sciences, University of California at Berkeley, Berkeley, California 94720, USA.
authors: Carla
authors: C
authors: Inouye
authors: Department of Biology, University of Osnabrück, 49076 Osnabrück, Germany.
authors: Changjiang
authors: C
authors: You
authors: Department of Biology, University of Osnabrück, 49076 Osnabrück, Germany.
authors: Jacob
authors: J
authors: Piehler
authors: Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037 USA.
authors: Peter G
authors: PG
authors: Schultz
authors: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA.
authors: Luke D
authors: LD
authors: Lavis
authors: Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 9HN, United Kingdom.
authors: Andrey
authors: A
authors: Revyakin
authors: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA.|Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA.|Li Ka Shing Center for Biomedical and Health Sciences, University of California at Berkeley, Berkeley, California 94720, USA.
authors: Robert
authors: R
authors: Tjian
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D008857:Microscopy, Interference; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology*; D012319:RNA Polymerase II / Q000378:metabolism; D017384:Sequence Deletion; D013997:Time Factors; D019473:Transcription Factors, TFII / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology*
chemical_list: D019473:Transcription Factors, TFII; D012319:RNA Polymerase II
keywords: dynamic analysis; fluorescence imaging; in vitro reconstitution; preinitiation complex; single-molecule; transcription