Transcriptional regulation of gene expression is a complex task, critical for growth and survival, whether as part of the developmental process from the fertilized egg, or when adapting to changing environmental conditions. The transcription initiation step is arguably the most regulated step in gene transcription, as fine tuning both its rate and synchrony can serve as a key control point to produce organism-wide changes in gene expression profiles in response to developmental and environmental cues. Not surprisingly, the complexity of gene regulatory circuitries is paralleled by the size and complexity of the molecular players involved in transcription initiation. Over the past 30 years, biochemistry, molecular genetics, and in vivo studies have uncovered most, if not all, of the central components of the transcriptional apparatus. However, a mechanistic understanding of gene expression in humans poses a formidable challenge and lags dramatically behind. A major obstacle is that the transcriptional machinery comprises more than 100 individual polypeptides that operate as a huge and dynamic assemblage made up of functionally distinct multi-subunit complexes, many of them only accessible from endogenous sources. We are using single particle EM reconstruction to characterize the architecture, dynamics and interactions of large human complexes essential for gene regulation. 3D Cryo-EM reconstruction is a technique ideally suited to this task, as it requires limited amounts of material, is optimal to study very large assemblies, and is has the potential to detect and characterize conformational flexibility. The latter is a property that may prove critical to be able to describe the functional plasticity required in transcriptional complexes like TFIID, an essential hub in this process that needs to bind to different DNA core promoters and integrate the input from a large variety of transcriptional activators and cofactors. The significance to human health of a fundamental understanding of how gene transcription is switched on and off cannot be overstated. Such significance is highlighted by the fact that development of various cancers is accompanied by alterations in gene expression leading to various aspects of the disease, and by the discovery of induced pluripotent stem cells, where the expression of 3-4 global transcriptional activators is sufficient to introduce gene expression changes needed to transition into a pluripotent state. By understanding the protein structures necessary for gene activation, we will guide future research into the development of novel treatments that target the control of gene expression mediating cell growth, neoplasia, metastasis, and angiogenesis in humans or those aimed at facilitating the transition of induced pluripotent stem cells research into the clinic.

Public Health Relevance

Transcription, the process of copying DNA genes into messenger RNA, is an essential step for determining the amount of protein a cell ultimately makes, and its regulation is critical both during development or when adapting to changing environmental conditions. A major control point is the initiation of transcription, which involves a molecular machinery of over 100 different proteins. We are using state of the art electron microscopy and image analysis to characterize this machinery and gain mechanistic understanding that will be critical for the development of novel treatments that target the control of gene expression mediating cell growth, neoplasia, metastasis, and angiogenesis in humans, or for research that leads to clinical uses of pluripotent stem cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063072-11
Application #
8734436
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
2001-05-01
Project End
2017-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
11
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Miscellaneous
Type
Organized Research Units
DUNS #
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Nogales, Eva; Louder, Robert K; He, Yuan (2016) Cryo-EM in the study of challenging systems: the human transcription pre-initiation complex. Curr Opin Struct Biol 40:120-127
He, Yuan; Yan, Chunli; Fang, Jie et al. (2016) Near-atomic resolution visualization of human transcription promoter opening. Nature 533:359-65
Louder, Robert K; He, Yuan; López-Blanco, José Ramón et al. (2016) Structure of promoter-bound TFIID and model of human pre-initiation complex assembly. Nature 531:604-9
Nogales, Eva (2016) The development of cryo-EM into a mainstream structural biology technique. Nat Methods 13:24-7
Nogales, Eva; Scheres, Sjors H W (2015) Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity. Mol Cell 58:677-89
Zhang, Elisa T; He, Yuan; Grob, Patricia et al. (2015) Architecture of the human XPC DNA repair and stem cell coactivator complex. Proc Natl Acad Sci U S A 112:14817-22
Ciferri, Claudio; Lander, Gabriel C; Nogales, Eva (2015) Protein domain mapping by internal labeling and single particle electron microscopy. J Struct Biol 192:159-62
He, Yuan; Fang, Jie; Taatjes, Dylan J et al. (2013) Structural visualization of key steps in human transcription initiation. Nature 495:481-6
Kassube, Susanne A; Jinek, Martin; Fang, Jie et al. (2013) Structural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5. Nat Struct Mol Biol 20:892-9
Cianfrocco, Michael A; Nogales, Eva (2013) Regulatory interplay between TFIID's conformational transitions and its modular interaction with core promoter DNA. Transcription 4:120-6

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