Structure, assembly and dynamics of macromolecular complexes by single particle cryo-electron microscopy

Abstract

BackgroundProteins in their majority act rarely as single entities. Multisubunit macromolecular complexes are the actors in most of the cellular processes. These nanomachines are hold together by weak protein-protein interactions and undergo functionally important conformational changes. TFIID is such a multiprotein complex acting in eukaryotic transcription initiation. This complex is first to be recruited to the promoter of the genes and triggers the formation of the transcription preinitiation complex involving RNA polymerase II which leads to gene transcription. The exact role of TFIID in this process is not yet understood.MethodsLast generation electron microscopes, improved data collection and new image analysis tools made it possible to obtain structural information of biological molecules at atomic resolution. Cryo-electron microscopy of vitrified samples visualizes proteins in a fully hydrated, close to native state. Molecular images are recorded at liquid nitrogen temperature in low electron dose conditions to reduce radiation damage. Digital image analysis of these noisy images aims at improving the signal-to-noise ratio, at separating distinct molecular views and at reconstructing a three-dimensional model of the biological particle.ResultsUsing these methods we showed the early events of an activated transcription initiation process. We explored the interaction of the TFIID coactivator with the yeast Rap1 activator, the transcription factor TFIIA and the promoter DNA. We demonstrated that TFIID serves as an assembly platform for transient protein-protein interactions, which are essential for transcription initiation.ConclusionsRecent developments in electron microscopy have provided new insights into the structural organization and the dynamic reorganization of large macromolecular complexes. Examples of near-atomic resolutions exist but the molecular flexibility of macromolecular complexes remains the limiting factor in most case. Electron microscopy has the potential to provide both structural and dynamic information of biological assemblies in order to understand the molecular mechanisms of their functions.

Hemocyanins of Muricidae: New ‘Insights’ Unravel an Additional Highly Hydrophilic 800 kDa Mass Within the Molecule

“…It is a well-known phenomenon of single particle analyses not to resolve highly flexible parts of molecules because this method is founded on image averages of the same views of molecules. Structural flexibilities will be averaged out when merged to a 3D model (Orlova and Saibil 2010 ; Durand et al 2013 ).…”

Protein Promiscuity in H2O2Signaling

“…As cryo-electron microscopy (cryo-EM) has not the same restrictions of conformational homogeneity due to the computational sorting and filtering of sample images, it lends itself well to the structural study of multiprotein complexes (80,164,165). In addition to nonnative complexes, cryo-EM is also especially well suited to analyze large native macromolecular assemblies of subunits.…”

dTAF10- and dTAF10b-Containing Complexes Are Required for Ecdysone-Driven Larval-Pupal Morphogenesis in Drosophila melanogaster

“…In vivo studies highlight that both functional Drosophila and human core TFIID complexes contain dTAF4, dTAF5, dTAF6, dTAF9, and dTAF12 in their central regions [ 8 ]. The TAF8-TAF10 heterodimer is present in one copy in the human TFIID core complex (called 7TAF) [ 9 – 11 ]. After the binding of TAF8–TAF10 to the TAF4, TAF5, TAF6, TAF9, and TAF12-containing TFIID core complex, conformational change occurs inside the TFIID [ 9 – 11 ].…”

“…The TAF8-TAF10 heterodimer is present in one copy in the human TFIID core complex (called 7TAF) [ 9 – 11 ]. After the binding of TAF8–TAF10 to the TAF4, TAF5, TAF6, TAF9, and TAF12-containing TFIID core complex, conformational change occurs inside the TFIID [ 9 – 11 ]. Interestingly, both TAF10- and TAF2-lacking TFIID complexes have been described from human cells [ 12 – 14 ].…”