Special Biochemistry Seminar

Snapshots of Transcription Initiation by Cryo-EM

Low-dose electron microscopy of cryo-preserved individual biomolecules (single-particle cryo-EM) is a powerful tool for obtaining information about the structure and dynamics of large macromolecular assemblies. Acquiring images with low dose reduces radiation damage, preserves atomic structural details, but results in low signal-to-noise ratio of the individual images. The projection directions of the two-dimensional images are random and unknown. The grand challenge is to achieve the precise three-dimensional (3D) alignment of many (tens of thousands) noisy projection images, which may then be combined to obtain a faithful 3D map. I will describe a new unbiased single-particle 3D reconstruction approach relying on probabilistic inference and stochastic optimization. A sparse continuous distributions of weights that relates the continuous distribution of orientation and CTF parameters of every image to the 3D reconstruction is determined. In a single step, without any user intervention or input other than the noisy individual particle images and the microscope-dependent parameters associated with them, a high-resolution (< 10 Å) map can be generated.

I will also describe the protein density and arrangement of subunits of a complete, 32-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC), determined by means of cryogenic electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs) and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft.