Escherichia coli RNA polymerase core and holoenzyme structures

Multisubunit RNA polymerase is an essential enzyme for regulated gene expression. Here we report two Escherichia coli RNA polymerase structures: an 11.0 A structure of the core RNA polymerase and a 9.5 A structure of the sigma(70) holoenzyme. Both structures were obtained by cryo-electron microscopy and angular reconstitution. Core RNA polymerase exists in an open conformation. Extensive conformational changes occur between the core and the holoenzyme forms of the RNA polymerase, which are largely associated with movements in ss'. All common RNA polymerase subunits (alpha(2), ss, ss') could be localized in both structures, thus suggesting the position of sigma(70) in the holoenzyme.     

A rapid method for the purification of RNA polymerase holoenzyme from Escherichia coli

A method is described for the rapid purification of RNA polymerase holoenzyme from small amounts of Escherichia coli cells. Chromatography of a crude extract on a single-stranded DNA agarose column followed by gel filtration chromatography gave 95% pure holoenzyme. The enzyme had kinetic characteristics on T7 DNA identical to those of RNA polymerase purified by other more laborious procedures.     

Separation of Escherichia coli RNA polymerase sigma-70 holoenzyme from core enzyme on heparin-Sepharose columns

A method is described for the rapid purification of DNA-dependent RNA polymerase sigma-70 holoenzyme from Escherichia coli. The essential step in this protocol involves the differential elution of sigma-70 holoenzyme from core polymerase on a heparin-Sepharose column. Using a linear gradient of KCl, holoenzyme was found to elute at 0.25 M whereas core polymerase eluted at 0.35 M. From 20 g of cells, up to 1 mg of RNA polymerase holoenzyme could be isolated in two days. The preparations were greater than 95% pure with respect to protein, and saturated with the sigma subunit.     

DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits.

DNA polymerase III holoenzyme has been purified from Escherichia coli HMS-83, using, as an assay, the conversion of coliphage G4 single-stranded DNA to the duplex replicative form. The holoenzyme consists of at least four different subunits: alpha, beta, gamma, and delta of 140,000, 40,000, 52,000, and 32,000 daltons, respectively. The alpha subunit is DNA polymerase III, the dnaE gene product. The holoenzyme has been resolved by phosphocellulose chromatography into an alpha - gamma - delta complex and a subunit beta (copolymerase III*); neither possesses detectable activity in the G4 system but together reconstitute holoenzyme-like activity. The alpha - gamma - delta complex has been further resolved to yield a gamma - delta complex which reconstitutes alpha - gamma - delta activity when added to DNA polymerase III. The gamma - delta complex contains a product of the dnaZ gene and has been purified from a strain which contains a ColE1-dnaZ hybrid plasmid.     

Aggregation equilibria of Escherichia coli RNA polymerase: evidence for anion-linked conformational transitions in the protomers of core and holoenzyme

The aggregation equilibria of Escherichia coli RNA polymerase core and holoenzyme have been studied by velocity sedimentation as a function of [NaCl] both in the presence and in the absence of MgCl2. Effects of other anions (F- and I-), pH, and temperature have also been examined. Diffusion coefficients obtained by quasi-elastic light scattering (QLS) at high and low salt concentrations were used in conjunction with sedimentation coefficients under these conditions to obtain molecular weights of the protomer and aggregates of the core enzyme. At low salt concentration, core aggregates to a tetramer in the absence of MgCl2 and to an octamer in the presence of MgCl2. Some ambiguity exists in the interpretation of the sedimentation and QLS data for holoenzyme. The sedimentation results are consistent with the formation of dimers at low salt, both in the presence and in the absence of MgCl2. In all cases, equilibrium constants were calculated assuming a simple monomer--j-mer stoichiometry. These equilibrium constants are extremely sensitive functions of the concentration and type of monovalent anion. In Cl-, aggregation of both core and holoenzyme begins abruptly when the salt concentration is reduced below approximately 0.2 M (at a protein concentration of approximately 0.30 mg/mL); for core, substitution of I- for Cl- suppresses aggregation while F- enhances aggregation at a fixed anion concentration. No specific effect of monovalent cations (Na+, NH4+) is observed; Mg2+ has no effect on holoenzyme dimerization and has little effect on the salt range of core aggregation, though the stoichiometries of the core aggregates in the presence and absence of Mg2+ differ. Anion effects on these equilibria were modeled by assuming that a class of anion-binding sites on the protomer is not present in the aggregate, so that anion release accompanies aggregation. Analytical expressions for several models of the effect of anions on the aggregation equilibria were derived by using the method of binding polynomials. The salt dependence of the aggregation equilibria in the absence of Mg2+ appears inconsistent with a model in which the anion-binding sites on the protomer are independent (noncooperative), but it is well described by a model in which anion binding to the protomers occurs in a completely cooperative manner. The molecular basis of this apparent cooperative effect of anions on the aggregation equilibria is proposed to be an allosteric effect of anions on conformational equilibria of the protomers of core polymerase and the holoenzyme. Implications of such a salt-dependent conformational transition for the DNA-binding interactions of the enzyme are considered.     

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

Escherichia coli RNA polymerase holoenzyme forms two-dimensional crystals when adsorbed to positively charged lipid layers at the air/water interface. Adsorption of the protein is driven by electrostatic interactions between the positively charged lipid surface and the polymerase molecule, which has a net negative charge. Crystallization is dependent on the adsorption and concentration of RNA polymerase on fluid lipid surfaces. Image analysis of electron micrographs of crystals in negative stain, which diffract to 30 A resolution, shows irregularly shaped protein densities about 100 x 160 A, consistent with the dimensions of single polymerase molecules.     

Binding of Escherichia coli RNA polymerase to T7 DNA. Displacement of holoenzyme from promoter complexes by heparin.

Escherichia coli RNA polymerase holoenzyme bound to promoter sites on T7 DNA is attacked and inactivated by the polyanion heparin. The highly stable RNA polymerase-T7 DNA complex formed at the major T7 A1 promoter can be completely inactivated by treatment with heparin, as shown by monitoring the loss of activity of such complexes, and by gel electrophoresis of the RNA products transcribed. The rate of this inactivation is much faster than the rate of dissociation of RNA polymerase from promoter complexes, and thus represents a direct attack of heparin on the polymerase molecule bound at promoter A1. Experiments employing the nitrocellulose filter binding technique suggest that heparin inactivates E. coli RNA polymerase when bound to T7 DNA by directly displacing the enzyme from the DNA. RNA polymerase bound at a minor T7 promoter (promoter C) is much less sensitive to heparin attack than enzyme bound at promoter A1. Thus, the rate of inactivation of RNA polymerase-T7 DNA complexes by heparin is dependent upon the structure of the promoter involved even though the inhibitor binds to a site on the enzyme molecule.     

Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme

Escherichia coli DNA polymerase III holoenzyme was used to synthesize double-stranded DNA from M13 single-stranded DNA hybridized to a phosphorylated synthetic oligodeoxynucleotide containing a nucleotide substitution. The resulting DNA was transfected into E. coli JM101 without further treatment. Sequence analysis of randomly chosen phage clones revealed that the efficiency of mutagenesis was nearly 50%, which is the theoretical maximum. Treatment with DNA ligase after DNA synthesis was not necessary to obtain high efficiency of mutagenesis. Thus, use of DNA polymerase III holoenzyme provides a simple and efficient procedure for site-directed mutagenesis.