Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes

被引:39
作者
Baek, Inwha [1 ]
Friedman, Larry J. [2 ]
Gelles, Jeff [2 ]
Buratowski, Stephen [1 ]
机构
[1] Harvard Med Sch, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USA
[2] Brandeis Univ, Dept Biochem, Waltham, MA 02454 USA
关键词
TRANSCRIPTION INITIATION; PREINITIATION COMPLEX; YEAST TRANSCRIPTION; PROMOTER; TFIIH; PURIFICATION; PROTEINS; HOLOENZYME; MECHANISM; BASAL;
D O I
10.1016/j.molcel.2021.07.025
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can preassemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.
引用
收藏
页码:3576 / +
页数:20
相关论文
共 69 条
[61]   The general transcription machinery and general cofactors [J].
Thomas, Mary C. ;
Chiang, Cheng-Ming .
CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, 2006, 41 (03) :105-178
[62]  
THOMPSON NE, 1990, J BIOL CHEM, V265, P7069
[63]   TFIIH generates a six-base-pair open complex during RNAP II transcription initiation and start-site scanning [J].
Tomko, Eric J. ;
Fishburn, James ;
Hahn, Steven ;
Galburt, Eric A. .
NATURE STRUCTURAL & MOLECULAR BIOLOGY, 2017, 24 (12) :1139-+
[64]   Mechanisms and Evolution of Control Logic in Prokaryotic Transcriptional Regulation [J].
van Hijum, Sacha A. F. T. ;
Medema, Marnix H. ;
Kuipers, Oscar P. .
MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, 2009, 73 (03) :481-+
[65]   Conservation between the RNA Polymerase I, II, and III Transcription Initiation Machineries [J].
Vannini, Alessandro ;
Cramer, Patrick .
MOLECULAR CELL, 2012, 45 (04) :439-446
[66]   PREPARATION AND CHARACTERIZATION OF YEAST NUCLEAR EXTRACTS FOR EFFICIENT RNA POLYMERASE-B (II)-DEPENDENT TRANSCRIPTION INVITRO [J].
VERDIER, JM ;
STALDER, R ;
ROBERGE, M ;
AMATI, B ;
SENTENAC, A ;
GASSER, SM .
NUCLEIC ACIDS RESEARCH, 1990, 18 (23) :7033-7039
[67]   A transcription reinitiation intermediate that is stabilized by activator [J].
Yudkovsky, N ;
Ranish, JA ;
Hahn, S .
NATURE, 2000, 408 (6809) :225-229
[68]   Measuring dynamics of eukaryotic transcription initiation: Challenges, insights and opportunities [J].
Zhang, Zhengjian ;
Tjian, Robert .
TRANSCRIPTION-AUSTIN, 2018, 9 (03) :159-165
[69]   Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis [J].
Zhang, Zhengjian ;
English, Brian P. ;
Grimm, Jonathan B. ;
Kazane, Stephanie A. ;
Hu, Wenxin ;
Tsai, Albert ;
Inouye, Carla ;
You, Changjiang ;
Piehler, Jacob ;
Schultz, Peter G. ;
Lavis, Luke D. ;
Revyakin, Andrey ;
Tjian, Robert .
GENES & DEVELOPMENT, 2016, 30 (18) :2106-2118