Professor Eva Nogales
Biochemistry and Molecular Biology
University of California @ Berkeley
Berkeley, CA

Monday, September 22, 2014
3:00 pm (CT)
3269 Beckman Institute

Abstract

Cryo-Electron Microscopy (cryo-EM) has emerged as an ideal technique for the structural characterization of challenging macromolecular assemblies. Recent technical breakthroughs have dramatically improved both the resolution obtainable and the capacity to describe coexisting states. We are using this methodology to gain fundamental insight into two essential and highly regulated processes in the life of the eukaryotic cell: microtubule dynamic instability and gene transcription initiation. Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice, and is inhibited by anticancer agents like Taxol. We have obtained new insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. Our analysis suggests how hydrolysis leads to conformational strain that would be released by bending during depolymerization. We are also characterizing the interaction of microtubules with proteins that regulate dynamic instability. Eukaryotic transcription initiation requires the assembly of general transcription factors into a pre-initiation complex (PIC) that ensures the accurate loading of RNA polymerase II (Pol II) at the transcription start site. We have used an in vitro reconstituted system to study the assembly of human TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE and TFIIH onto promoter DNA using cryo-EM. Our structural analysis provides pseudo-atomic models of the PIC and its engagement with DNA, before and after promoter opening, that allow us to propose a mechanistic model of how the action of the XPB ATPase in TFIIH melts the DNA.