Purification of RNA Polymerase Elongation Complexes for Cryoelectron Microscopy Investigation

Resumo

A procedure was developed for the preparation of a stalled elongation complex of Escherichia coli RNA polymerase with the nucleosome for investigation by cryo-electron microscopy. We purified the complex from the excess of free RNA polymerase and unproductive complexes on heparin resin and concentrated it on an affine monolayer formed by lipids bound to Ni ions. The use of affinity grids with an immobilized lipid monolayer helps to prevent aggregation of the particles on the grid surface. This technique can be used in the future to obtain a three-dimensional reconstruction of the EC+39 elongation complex.