Spatial arrangement of sigma-factor and core enzyme of Escherichia coli RNA polymerase. A neutron solution scattering study

By means of neutron solution scattering we determined the position and orientation of core enzyme and sigma-factor within the Escherichia coli RNA polymerase holoenzyme with the aim of improving existing models. The individual components, core enzyme (E) and sigma-factor (sigma), were highlighted by deuterium labeling and their center-to-center distances determined in the monomeric and the dimeric holoenzyme. The following distance parameters were obtained: dE1-sigma 1 = 8.6(+/- 1) nm, dE1-E2 = 11.5(+/- 1) nm, d sigma 1-sigma 2 = 12.0(+/- 0.7) nm, dE1-sigma 2 = 9(+/- 3) nm. Using a triangulation procedure the position of the sigma-factors, sigma 1 and sigma 2, were determined with respect to the mass center of the core enzyme molecules, E1 and E2, assuming a symmetrical arrangement of the holoenzyme molecules in the dimer (C2 symmetry). In addition, the orientation of the sigma-factor with respect to core enzyme was estimated by means of model calculations. The obtained model of holoenzyme depicts the sigma-factor as buried in a groove of core enzyme, probably between the large subunits beta' and beta.     

DNA-dependent RNA polymerase of Escherichia coli induces bending or an increased flexibility of DNA by specific complex formation.

Escherichia coli RNA polymerase is shown to induce bending or an increased flexibility of the promoter DNA. This is a specific effect of holoenzyme (core enzyme and sigma-factor). The centre of the flexibility is 3 bp upstream of the initiation point of RNA synthesis. This flexibility or bending is maintained during RNA synthesis by core enzyme.     

Deuterium incorporation into Escherichia coli proteins. A neutron-scattering study of DNA-dependent RNA polymerase

Neutron small-angle scattering studies of single protein subunits in a protein-DNA complex require the adjustment of the neutron scattering-length densities of protein and DNA, which is attainable by specific deuteration of the protein. The neutron scattering densities of unlabelled DNA and DNA-dependent RNA polymerase of Escherichia coli match when RNA polymerase is isolated from cells grown in a medium containing 46% D2O and unlabelled glucose as carbon source. Their contrasts vanish simultaneously in a dialysis buffer containing 65% D2O. An expression was evaluated which allows the calculation of the degree of deuteration and match point of any E. coli protein from the D2O content of the growth medium, taking the 2H incorporation into RNA polymerase amino acids to be representative for all amino acids in E. coli proteins. The small-angle scattering results, on which the calculation of the degree of deuteration is based, were confirmed by mass spectrometric measurements.     

Binding and transcription of simian virus 40 DNA by DNA-dependent RNA polymerase from Escherichia coli.

Supercoiled simian virus 40 was transcribed more efficiently than nonsupercoiled DNA. The effect was increased from two- to fivefold by the addition of rifampin with triphosphates. The number and locations of polymerase binding sites with respect to Hin II-III restriction fragments were determined. The total number of binding sites was nine, as determined by UV difference spectroscopy. The locations of these binding sites were on the A, B, D, E, F, and G fragments, as determined by gel electrophoresis. The number of sites was the same for both supercoiled and relaxed or Hin II-III-digested DNA, and the point of saturation of supercoiled DNA by polymerase remained the same with increasing concentrations of rifampin from 0 to 8 microgram/ml.     

The structure of the zinc sites of Escherichia coli DNA-dependent RNA polymerase.

X-ray absorption spectroscopy is ideally suited for the investigation of the electronic structure and the local environment (approximately 5 A) of specific atoms in biomolecules. While the edge region provides information about the valence state of the absorbing atom, the chemical identity of neighboring atoms, and the coordination geometry, the extended x-ray absorption fine structure region contains information about the number and average distance of neighboring atoms and their relative disorder. The development of sensitive detection methods has allowed studies using near physiological concentrations (as low as approximately 100 microM). RNA polymerase from Escherichia coli contains two zinc atoms: one tightly bound in the beta' subunit, the subunit that participates in template binding, and the other loosely bound in the beta subunit, the subunit that participates in substrate binding. X-ray absorption studies of these zinc sites in the native protein and of the zinc site in the beta' subunit after removal of the zinc in the beta subunit site by p-(hydroxymercuri)benzenesulfonate (Giedroc, D. P., and Coleman, J. E. (1986) Biochemistry 25, 4969-4978) indicate that both zinc sites have octahedral coordination. The zinc in the beta' subunit site has four sulfur ligands at an average distance of 2.36 +/- 0.02 A and two oxygen (or nitrogen) ligands at an average distance of 2.23 +/- 0.02 A. The beta subunit zinc site has five sulfur ligands at an average distance of 2.38 +/- 0.01 A and one histidine nitrogen ligand at 2.14 +/- 0.02 A. These results are in general agreement with earlier biochemical and spectroscopic studies.     

Synthesis of complementary RNA on RNA templates using the DNA-dependent RNA polymerase of Escherichia coli.

It is shown that the DNA-dependent RNA polymerase of Escherichia coli can synthesize complementary RNA (cRNA) directly on rRNA and mRNA templates. Synthesis occurred preferentially in the presence of Mn2+ and at relatively high substrate and enzyme concentrations. No primer was required, and addition of oligo-U to a mRNA-dependent reaction gave no marked stimulation. Sedimentation analysis of cRNA made on different templates indicated that the products were mainly 2-4 S, but a fraction of the product was larger. Fingerprints of 32P-labelled cRNA made on 5 S rRNA and 18 S rRNA indicated that the complexity of the cRNAs was related to the size of the template, suggesting that a substantial portion of the templates were copied. This reaction provides a simple method for preparing cRNA of high specific activity for use in hybridisation studies, and possibly in sequence analysis. 32P-labelled cRNA made on 18 S and 28 S rRNA was a sensitive hybridisation probe for detection of the specific fragments of mouse DNA containing the rRNA genes.     

Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase.

An improved purification procedure is described for the sigma subunit of escherichia coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70, and finally Ultragel AcA44. The sigma factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical--physical properties of sigma are presented. A molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of sigma have been determined. The isoelectric focusing properties of sigma are presented. Denaturation--renaturation studies indicate that sigma is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000-dalton fragment is generated from sigma by mild trypsin treatment.     

Spectroscopic analysis of the interaction of Escherichia coli DNA-dependent RNA polymerase with T7 DNA and synthetic polynucleotides.

We have studied the circular dichroism and ultraviolet difference spectra of T7 bacteriophage DNA and various synthetic polynucleotides upon addition of Escherichia coli RNA polymerase. When RNA polymerase binds nonspecifically to T7 DNA, the CD spectrum shows a decrease in the maximum at 272 but no detectable changes in other regions of the spectrum. This CD change can be compared with those associated with known conformational changes in DNA. Nonspecific binding to RNA polymerase leads to an increase in the winding angle, theta, in T7 DNA. The CD and UV difference spectra for poly[d(A-T)] at 4 degrees C show similar effects. At 25 degrees C, binding of RNA polymerase to poly[d(A-T)] leads to hyperchromicity at 263 nm and to significant changes in CD. These effects are consistent with an opening of the double helix, i.e. melting of a short region of the DNA. The hyperchromicity observed at 263 nm for poly[d(A-T)] is used to determine the number of base pairs disrupted in the binding of RNA polymerase holoenzyme. The melting effect involves about 10 base pairs/RNA polymerase molecule. Changes in the CD of poly(dT) and poly(dA) on binding to RNA polymerase suggest an unstacking of the bases with a change in the backbone conformation. This is further confirmed by the UV difference spectra. We also show direct evidence for differences in the template binding site between holo- and core enzyme, presumably induced by the sigma subunit. By titration of the enzyme with poly(dT) the physical site size of RNA polymerase on single-stranded DNA is approximately equal to 30 bases for both holo- and core enzyme. Titration of poly[d(A-T)] with polymerase places the figure at approximately equal to 28 base pairs for double-stranded DNA.     

Inhibition of DNA-dependent RNA polymerase from Escherichia coli by pyran copolymer

Pyran copolymer, a potent inhibitor of DNA-dependent RNA polymerase from Escherichia coli, prevented polyribonucleotide synthesis by blocking both the initiation and elongation steps. The inhibition was noncompetitive with respect to template and nucleotide triphosphate substrates. Template binding and the stability of the nascent RNA chain were not affected by the inhibitor.     

1H n.m.r. of the DNA-dependent RNA polymerase from Escherichia coli

The ¹H n.m.r. study of the DNA-dependent RNA polymerase from Escherichia coli has revealed that the holoenzyme (ββ′α₂σ) displays two mobile regions: one, observable also in the core enzyme (ββ′α₂), is characterized by basic amino acids and its appearance and form depend on ionic strength; the other, specific to the holoenzyme, is characterized by threonine residues and its appearance does not depend on ionic strength.     

A small angle X-ray study of the elongation factor complex Tu . GDP from Escherichia coli.

The protein elongation factor complex Tu. GDP from Escherichia coli was investigated in the presence of 0.01 mM GDP using the small-angle X-ray method. The overall shape and the molecular parameters of the Tu . GDP complex were determined using a least-squares method where the experimental data were used directly without desmearing. The best fit to the experimental data was obtained assuming the molecule to be an ellipsoid of revolution with the semiaxes A = B = 4.08 nm, and C = 1.18nm. Determination of the molecular weight gave the result Mr = 46 000, which corresponds to a water content equal to 26% (by weight).