Hybrid electron microscopy-FRET imaging localizes the dynamical C-terminus of Tfg2 in RNA polymerase II-TFIIF with nanometer precision
DC Element | Wert | Sprache |
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dc.contributor.author | Chang, Jen-Wei | |
dc.contributor.author | Wu, Yi-Min | |
dc.contributor.author | Chen, Zi-Yun | |
dc.contributor.author | Huang, Shih-Hsin | |
dc.contributor.author | Wang, Chun-Hsiung | |
dc.contributor.author | Wu, Pei-lun | |
dc.contributor.author | Weng, Yi-ping | |
dc.contributor.author | You, Changjiang | |
dc.contributor.author | Piehler, Jacob | |
dc.contributor.author | Chang, Wei-hau | |
dc.date.accessioned | 2021-12-23T16:16:36Z | - |
dc.date.available | 2021-12-23T16:16:36Z | - |
dc.date.issued | 2013 | |
dc.identifier.issn | 10478477 | |
dc.identifier.uri | https://osnascholar.ub.uni-osnabrueck.de/handle/unios/11952 | - |
dc.description.abstract | TFIIF-a general transcription factor comprising two conserved subunits can associate with RNA polymerase II (RNAPII) tightly to regulate the synthesis of messenger RNA in eukaryotes. Herein, a hybrid method that combines electron microscopy (EM) and Forster resonance energy transfer (FRET) is described and used to localize the C-terminus of the second TFIIF subunit (Tfg2) in the architecture of RNAPII-TFIIF. In the first stage, a poly-histidine tag appended to the Tfg2 C-terminus was labeled with nickel-NTA nanogold and a seven-step single particle EM protocol was devised to obtain the region accessible by the nanogold in 3D, suggesting the Tfg2 C-terminus is proximal to the clamp of RNAPII. Next, the C-termini of the Rpb2 and the Rpb4 subunits of RNAPII, adjacent to the clamp, were selected for placing FRET satellites to enable the nano-positioning (NP) analysis, by which the localization precision was improved such that the Tfg2 C-terminus was found to dwell on the clamp ridge but could move to the clamp top during transcription. Because the tag receptive to the EM or FRET probes can be readily introduced to any protein subunit, this hybrid approach is generally applicable to complement cryo-EM study of many protein complexes to nanometer precision. (c) 2013 Elsevier Inc. All rights reserved. | |
dc.description.sponsorship | NSCMinistry of Science and Technology, Taiwan; Academia Sinica Thematic Projects, Nanoscience Program; National Science Council of TaiwanMinistry of Science and Technology, Taiwan; The authors thank Prof. Jens Michaelis for the NPS software (University of Ulm, Germany); Dr. Joan Chen (IBMS, Academia Sinica, Taiwan) for providing the human CaM II clone. WHC has benefited from former Chang lab members: Tommy Setiawan for screening in-gel conditions, Dr. Yen-Chen Lin for CaM mutagenesis, and Dr. Chin-Yu Chen for helpful discussions on poly-histidine tag. Dr. Yi-min Wu has been supported by an NSC postdoctoral fellowship. Financial support to WHC is from Academia Sinica Thematic Projects, Nanoscience Program, and National Science Council of Taiwan. | |
dc.language.iso | en | |
dc.publisher | ACADEMIC PRESS INC ELSEVIER SCIENCE | |
dc.relation.ispartof | JOURNAL OF STRUCTURAL BIOLOGY | |
dc.subject | ANGSTROM RESOLUTION | |
dc.subject | Bio-conjugation | |
dc.subject | Biochemistry & Molecular Biology | |
dc.subject | Biophysics | |
dc.subject | Cell Biology | |
dc.subject | CRYO-EM | |
dc.subject | EUKARYOTIC TRANSCRIPTION | |
dc.subject | FATTY-ACID SYNTHASE | |
dc.subject | HISTIDINE-TAGGED PROTEINS | |
dc.subject | INITIATION COMPLEX | |
dc.subject | Malleable protein | |
dc.subject | Nano-probe | |
dc.subject | PREINITIATION COMPLEX | |
dc.subject | PROMOTER DNA | |
dc.subject | STRUCTURAL BASIS | |
dc.subject | Transcription | |
dc.subject | TRANSCRIPTION FACTOR TFIIF | |
dc.title | Hybrid electron microscopy-FRET imaging localizes the dynamical C-terminus of Tfg2 in RNA polymerase II-TFIIF with nanometer precision | |
dc.type | journal article | |
dc.identifier.doi | 10.1016/j.jsb.2013.05.015 | |
dc.identifier.isi | ISI:000325675000007 | |
dc.description.volume | 184 | |
dc.description.issue | 1 | |
dc.description.startpage | 52 | |
dc.description.endpage | 62 | |
dc.contributor.orcid | 0000-0002-2612-850X | |
dc.contributor.orcid | 0000-0002-7839-6397 | |
dc.contributor.orcid | 0000-0003-1023-118X | |
dc.contributor.researcherid | F-1965-2015 | |
dc.contributor.researcherid | L-3901-2014 | |
dc.contributor.researcherid | M-8764-2016 | |
dc.identifier.eissn | 10958657 | |
dc.publisher.place | 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA | |
dcterms.isPartOf.abbreviation | J. Struct. Biol. | |
crisitem.author.dept | Sonderforschungsbereich 944: Physiologie und Dynamik zellulärer Mikrokompartimente | - |
crisitem.author.dept | FB 05 - Biologie/Chemie | - |
crisitem.author.deptid | organisation19 | - |
crisitem.author.deptid | fb05 | - |
crisitem.author.orcid | 0000-0002-7839-6397 | - |
crisitem.author.orcid | 0000-0002-2143-2270 | - |
crisitem.author.parentorg | FB 05 - Biologie/Chemie | - |
crisitem.author.parentorg | Universität Osnabrück | - |
crisitem.author.grandparentorg | Universität Osnabrück | - |
crisitem.author.netid | YoCh745 | - |
crisitem.author.netid | PiJa938 | - |
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