Two-dimensional crystals of Escherichia coli RNA polymerase holoenzyme on positively charged lipid layers

Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals.

“…This structure was proposed to act as a clamp preventing the template from dissociating from the enzyme after each polymerization cycle thus allowing the processivity of the reaction. This DNA-binding groove would hide the probably positively charged residues from the outer surface in keeping with the observation that RNA polymerases do not bind to negatively charged lipids (Darst et al, 1988;Schultz et al, 1990a). The apical region, which interacts with the positively charged lipids, could possibly repel DNA and orient the enzyme so as to allow the DNA to interact with the groove.…”

“…Two-dimensional crystallization Two-dimensional crystallization of biological macromolecules using either charged (Darst et al, 1988(Darst et al, , 1989(Darst et al, , 1991Schultz et al, 1990a) or ligand exposing (Uzgiris and Kornberg, 1983;Ribi et al, 1987;Lebeau et al, 1990; Mosser et al, 1991) lipid layers has grown to become a powerful tool for electron microscopic studies of soluble proteins. These studies have shown that proteins are concentrated at the lipid -solvent interface upon interaction with the lipids and, in some cases, it has been shown that they are preferentially oriented (Schultz et al, 1990a (Darst et al, 1989(Darst et al, , 1990(Darst et al, , 1991.…”

“…Recent electron microscopic examination of twodimensional crystals of E. coli RNA polymerase (Darst et al, 1988) and yeast RNA polymerases I (Schultz et al, 1990a) and II (Edwards et al, 1990) grown on charged lipid layers shed some light on their complex molecular arrangement. To interpret the projection map of the crystals formed by the eukaryotic class A enzyme, we have determined the three-dimensional structure of negatively stained crystals, tilted at various observation angles, up to a resolution of -3 nm.…”

How can biochemical reactions within cells differ from those in test tubes?

“…Enhancement of equilibrium self-and/or hetero-association of adsorbed Proteins that do not self-associate in solution form 2D crystals when macromolecules (Minton, 1995). nonspecifically adsorbed to surfaces (Darst et al, 1988).…”

Protein Crystallization on Lipid Layers and Structure Determination of the RNA Polymerase II Transcription Initiation Complex

“…Although effective, this approach suffers both from limitation to available protein-ligand pairs and from the demand for lipid-ligand synthesis. More convenient and general approaches include the use of lipidadaptors, such as metal-lipids for binding oligohistidine-tagged proteins (10) and the use of uniformly charged lipid layers, for binding almost any protein through electrostatic interactions (11). Whereas binding to lipid-ligands and lipid-adaptors is not very dependent on solution conditions, binding through electrostatic interactions can only occur within a narrow range of ionic strength and would be expected to be pH-dependent as well.…”