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

Two-dimensional crystals of yeast RNA polymerase I dimers were obtained upon interaction with positively charged lipid layers. A three-dimensional surface model of the enzyme was determined by analyzing tilted crystalline areas and by taking advantage of the non-crystallographic internal symmetry of the dimer to correct for the missing viewing directions. The structure shows, at approximately 3 nm resolution, an irregularly shaped molecule 11 nm x 11 nm x 15 nm in size characterized by a 3 nm wide and 10 nm long groove which constitutes a putative DNA binding site. The overall structure is similar to the Escherichia coli holo enzyme and the yeast RNA polymerase II delta 4/7 structures. The most remarkable structural feature is a finger-shaped stalk which partially occludes the entrance of the groove and forms a 2.5 nm wide channel. We discuss the possible location of the catalytic centre and of the carboxy-terminal region of the beta-like subunit in the channel. The interference of different DNA fragments with RNA polymerase dimerization and crystallization indicates the orientation of the template in the putative DNA binding groove.     

Electron microscopic study of yeast RNA polymerase A: Analysis of single molecular images

The structural features of the yeast DNA-dependent RNA polymerase A (I) were examined by Scanning Transmission Electron Microscopy. The enzyme was adsorbed in its monomeric form and negatively stained prior to digital image acquisition at low dose. The signal to noise ratio of single particle images was improved through averaging of a large number of previously aligned and partitioned images. Six classes of images were obtained reproducibly which corresponded to different projections of the enzyme. The enzyme structure was characterized by its elongated shape of 15.5 by 10.5 nm and by the presence of two curved arms which defined a longitudinal cleft. By analogy with theEscherichia coli enzyme, these arms could correspond to the two large subunits A135 and A190.     

Specific interaction and two-dimensional crystallization of histidine tagged yeast RNA polymerase I on nickel-chelating lipids.

Nickel-chelating lipid monolayers were used to generate two-dimensional crystals from yeast RNA polymerase I that was histidine-tagged on one of its subunits. The interaction of the enzyme with the spread lipid layers was found to be imidazole dependent, and the formation of two-dimensional crystals required small amounts of imidazole, probably to select the specific interaction of the engineered tag with the nickel. Two distinct preparations of RNA polymerase I tagged on different subunits yielded two different crystal forms, indicating that the position of the tag determines the crystallization process. The orientation of the enzyme in both crystal forms is correlated with the location of the tagged subunits in a three-dimensional model which shows that the tagged subunits are in contact with the lipid layer.     

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.     

Single crystals of bacteriophage T7 RNA polymerase

Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 x 0.3 x 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which is enzymatically active upon dissolution. These crystals belong to the monoclinic space group P2(1) and have unit cell parameters a = 114.5 A, b = 139.6 A, c = 125.7 A, and beta = 98.1 degrees. Self-rotation function studies indicate that there are three molecules per asymmetric unit. The crystals diffract to at least 3.0 A resolution. These are the first crystals of a DNA-dependent RNA polymerase suitable for high-resolution X-ray structure determination.     

Fluorescence resonance energy transfer studies on the proximity relationship between the intrinsic metal ion and substrate binding sites of Escherichia coli RNA polymerase

DNA-dependent RNA polymerase from Escherichia coli contains 2 mol of zinc/mol of holoenzyme (alpha 2 beta beta' sigma) with one zinc each in the beta and beta' subunits. A new method to substitute selectively the zinc in the beta subunit was developed by the inactivation of RNA polymerase with 0.25 M NaNO3, 1 M NaCl, 1 mM diaminocyclohexane tetraacetic acid, and 0.1 mM dithiothreitol followed by reconstitution with Co(II), Cd(II), or Cu(II). The hybrid Co-Zn, Cd-Zn, or Cu-Zn RNA polymerase thus obtained retains, respectively, 91, 88, and 50% enzyme activity of the reconstituted Zn-Zn RNA polymerase. Co-Zn RNA polymerase exhibits absorption maxima at 395 and 465 nm, and Cu-Zn RNA polymerase at 637 nm (epsilon = 815 M-1 cm-1). 1-Aminonaphthalene-5-sulfonic acid (AmNS) derivatives of ATP, UTP, and dinucleoside monophosphates (diNMPs), UpA or ApU, were synthesized with AmNS attached to NTP via a gamma-phosphoamidate bond or to diNMPs via a 5'-secondary amine linkage. Since the fluorescence emission maxima of (5'-AmNS)UpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP at 445, 464, and 464 nm, respectively, when excited at 340 nm, overlap the 465-nm absorption band of Co-Zn RNA polymerase, the spatial relationship between fluorescence substrate analogs and the intrinsic Co(II) in Co-Zn RNA polymerase was studied by fluorescence resonance energy transfer technique. The fluorescence of the initiator, (5'-AmNS)UpA, and elongator, (gamma-AmNS)UTP, of the RNA chain, was quenched 20.3 and 7.1%, by the addition of saturation concentration of Zn-Zn RNA polymerase, and 21.3 and 14.7%, respectively, by the addition of template, poly(dA-dT). The fluorescence of (5'-AmNS)UpA and (gamma-AmNS)UTP was quenched 81.8 and 80.6%, respectively, by the addition of the saturation concentration of Co-Zn RNA polymerase in the absence of template, and 82.7 and 82.9% in the presence of template. On the basis of respective Ro values of 21.3 and 21.9 A for the (5'-AmNS)UpA-Co and (gamma-AmNS)UTP-Co pairs, the distances from Co(II) to the initiation site and to the elongation site were calculated to be 17.4 and 17.5 A, respectively, in the absence and 17.2 and 17.4 A in the presence of template.     

Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals

A simple in vitro substitution method involving a sequential denaturation--reconstitution process was developed to substitute selectively one of the two intrinsic Zn ions in Escherichia coli RNA polymerase with Co, Mn, Ni, or Cu ion. The resultant metal hybrid Co-Zn, Mn-Zn, Ni-Zn, and Cu-Zn RNA polymerases possess 100, 100, 60, and 17% of the enzymatic activity of the reconstituted Zn-Zn enzyme, respectively. The substituted metal was found to be located in the beta subunit of the polymerase which contains the substrate binding site. The biochemical and physical properties of these metal-substituted polymerases were compared with those of the native Zn enzyme. Co-Zn and Ni-Zn core polymerases exhibit characteristic absorption spectra in the near-UV and visible region, while Mn-Zn and Cu-Zn enzymes do not. The Co-Zn enzyme shows two major peaks at 400 nm (epsilon = 3000) and 475 nm (epsilon = 2700), while the Ni-Zn enzyme exhibits a major peak at 462 nm (epsilon = 8000). The difference absorption spectrum of Ni-Zn core polymerase could be perturbed by the addition of substrate ATP but not by UTP in the absence of template and Mg(II) ion. These observations suggest that the substituted metal was located at the initiation site of the enzyme. The various metal hybrid enzymes do not differ appreciably in their abilities to incorporate noncomplementary nucleotide or deoxyribonucleotide into RNA product. It was found, however, that the difference in enzymatic activities of these metal hybrid enzymes resides at least partly in the initiation step of RNA synthesis.     

Inhibition of hepatic deoxyribonucleic acid-dependent ribonucleic acid polymerases by the exotoxin of Bacillus thuringiensis in comparison with the effects of α-amanitin and cordycepin

The action of Bacillus thuringiensis exotoxin, a structural analogue of ATP, on mouse liver DNA-dependent RNA polymerases was studied and its effects were compared with those of alpha-amanitin and cordycepin. (1) Administration of exotoxin in vivo caused a marked decrease in RNA polymerase activity of isolated nuclei at various concentrations of Mg(2+), Mn(2+) and (NH(4))(2)SO(4). A similar action was recorded after addition of exotoxin to isolated nuclei from control or exotoxin-treated mice. (2) Chromatographic separation of nuclear RNA polymerases from mice treated in vivo with exotoxin showed a drastic decrease of the peak of nucleoplasmic RNA polymerase, whereas the peak of nucleolar RNA polymerase remained unaltered. The same effect was observed after administration of alpha-amanitin in vivo, but cordycepin did not alter the relative amounts of the two main RNA polymerase peaks. (3) Administration of exotoxin in vivo did not alter the template activity of isolated DNA or chromatin tested with different fractions of RNA polymerase from control or exotoxin-treated mice. (4) Addition of exotoxin to isolated liver RNA polymerases inhibited both enzyme fractions. However, the alpha-amanitin-sensitive RNA polymerase was also 50-100-fold more sensitive to exotoxin inhibition than was the alpha-amanitin-insensitive RNA polymerase. Kinetic analysis indicated the exotoxin produces a competitive inhibition with ATP on the nucleolar enzyme, but a mixed type of inhibition with nucleoplasmic enzyme. The results obtained indicate that the B. thuringiensis exotoxin inhibits liver RNA synthesis by affecting nuclear RNA polymerases, showing a preferential inhibition of the nucleoplasmic alpha-amanitin-sensitive RNA polymerase.     

Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I.     

Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.     

W-reactivation of phage lambda in recF, recL, uvrA, and uvrB mutants of E. coli K-12.

Summary Assay conditions are described which permit detection of cryptic temperature sensitive RNA polymerases in vitro. RNA polymerase was prepared from fifteen different temperature sensitive mutants of Salmonella typhimurium chosen at random from a larger group isolated by localized mutagenesis and uridine suicide techniques. The dependence of enzyme activity on temperature, ionic strength and pH was studied in vitro. Assays at higher ionic strength (0.23 M) and temperature (50°C) distinguish three classes of mutants (Table 2). Activity of seven mutant RNA polymerases (called Class 1) under these conditions was 1% to 5% that of the parental RNA polymerase. Five mutant RNA polymerases (called Class 2) had 18% to 64% of the parental activity and three were not distinguishable from the parental enzyme under these conditions. Mixing experiments showed that the defect in Class 1 mutant enzymes is a property of the enzymes and not due to a diffusible inhibitor. In one case the lesion was shown to reside in the core enzyme. Class 1 mutant RNA polymerases were shown to be irreversibly inactivated during the assay at higher temperature and ionic strength. This suggests that the Class 1 enzymes may be more thermolabile than the wild type enzyme or may fail to be protected from thermal denaturation by formation of a ternary complex with template and product. We conclude that the method used to isolate these mutants (Young et al., 1976) and the assay described here (Table 2) are efficient ways to isolate and detect temperature sensitive RNA polymerase mutants of Salmonella typhimurium.     

Two-dimensional and epitaxial crystallization of a mutant form of yeast RNA polymerase II

A mutant form of yeast RNA polymerase II that lacks the fourth and seventh largest subunits, referred to as pol II delta 4/7, crystallized on positively charged lipid layers. Both single-layered (two-dimensional) crystals and several multi-layered crystal forms were obtained. The two-dimensional crystals, preserved in negative stain, diffracted strongly to about 1/20 A-1 and more weakly to 1/13 A-1 resolution. A projection map computed from averaged Fourier transforms revealed four pol II delta 4/7 complexes per unit cell and further revealed a cleft on the surface of the complex similar to that previously observed in the structure of Escherichia coli RNA polymerase. One of the multi-layered crystal forms, preserved in negative stain, diffracted strongly beyond 1/15 A-1 resolution. Coherent diffraction from the multi-layered crystal is indicative of protein-protein interactions between layers and ordering in the third dimension.