Development of RNA polymerase-promoter contacts during open complex formation.

We have charted the movements of E sigma 32 RNA polymerase at the heat-shock promoter PgroE throughout open complex formation, using hydroxyl radical footprinting. In combination with methylation protection and DNase I experiments, these data suggest the following model for open complex formation. E sigma 32 initially anchors itself in the upstream region of the promoter forming the first closed complex, RPC1; in this complex the enzyme makes backbone contacts in the -35 region of the promoter that are maintained throughout open complex formation. An isomerization follows resulting in a second closed complex, RPC2; in this complex the enzyme makes base-specific and backbone contacts in the -10 region that are almost identical to those found in the open complex. Thus, at the groE promoter, upstream contacts are established in RPC1 and downstream contacts in RPC2. A similar pattern of backbone contacts was obtained for E sigma 32 bound in the open complex at two additional heat-shock promoters, suggesting that the overall topology of holoenzyme in the open complex is similar regardless of sequence variations in the promoter.     

Kinetics of RNA polymerase-promoter complex formation: effects of nonspecific DNA-protein interactions.

The rates of formation of RNA polymerase-promoter open complexes at the galactose P2 and lactose UV5 promoters of E. coli were studied using polyacrylamide gels to separate the heparin-resistant complexes from unbound DNA. Both the apparent rate and extent of reaction at these promoters are inhibited at excess RNA polymerase. This inhibition, which can be relieved by the addition of non-promoter DNA, is interpreted to be the result of occlusion of the promoter site by nonspecifically bound polymerase. Additionally, biphasic kinetics are observed at both gal P2 and lac UV5, but not at the PR promoter of phage lambda. This behavior disappears when the concentration of RNA polymerase in the binding reaction is less than that of the promoter fragment. It is proposed that at excess enzyme nonspecifically bound polymerase molecules sliding along the DNA may "bump" closed complexes from the promoter site thereby reducing the rate of open complex formation. Kinetics mechanisms quantifying both the occlusion and bumping phenomena are presented.     

Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex

We have used systematic fluorescence resonance energy transfer and distance-constrained docking to define the three-dimensional structures of bacterial RNA polymerase holoenzyme and the bacterial RNA polymerase-promoter open complex in solution. The structures provide a framework for understanding sigma(70)-(RNA polymerase core), sigma(70)-DNA, and sigma(70)-RNA interactions. The positions of sigma(70) regions 1.2, 2, 3, and 4 are similar in holoenzyme and open complex. In contrast, the position of sigma(70) region 1.1 differs dramatically in holoenzyme and open complex. In holoenzyme, region 1.1 is located within the active-center cleft, apparently serving as a "molecular mimic" of DNA, but, in open complex, region 1.1 is located outside the active center cleft. The approach described here should be applicable to the analysis of other nanometer-scale complexes.     

On the promoter complex formation rate of E. coli RNA polymerases with T7 phage DNA.

Influence of ionic strength on the kinetics of the promoter complex formation between E. coli RNA polymerase and T7 phage DNA was investigated using a membrane filter assay. The enzyme-promoter association rate constant was determined. It varies from 10(9) to 3 x 10(7) M-1 sec-1 when the ionic strength is changed from zero to 0.15 M NaCl. Basing on the theoretical analysis of experimental data obtained the model for the promoter site selection assuming the enzyme sliding along the DNA is discussed.     

DNA sliding and loop formation by E. coli SMC complex: MukBEF

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.     

The kinetics of E. coli RNA polymerase.

Using an assay specific for chain elongation of E. coli RNA polymerase the kinetics of this propagation reaction have been studied. The kinetic behaviour is consistent woth the mathematical model formulated for this multisubstrate enzyme. The effect of increasing salt concentration on the kinetics of the reaction indicated that DNA unwinding is probably a necessary step in the propagation step, although this may not be the rate limiting step under all conditions.     

Visualization and quantitative analysis of complex formation between E. coli RNA polymerase and an rRNA promoter in vitro.

We have established conditions that stabilize the interaction between RNA polymerase and the rrnB P1 promoter in vitro. The requirements for quantitative complex formation are unusual for E. coli promoters: (1) The inclusion of a competitor is required to allow visualization of a specific footprint. (2) Low salt concentrations are necessary since complex formation is salt sensitive. (3) The addition of the initiating nucleotides ATP and CTP, resulting in a low rate of dinucleotide production, is required in order to prevent dissociation of the complexes. The complex has been examined using DNAase I footprinting and filter binding assays. It is characterized by a region protected from DNAase I cleavage that extends slightly upstream of the region protected by RNA polymerase in most E. coli promoters. We find that only one mole of active RNA polymerase is required per mole of promoter DNA in order to detect filter-bound complexes. Under the conditions measured, the rate of association of RNA polymerase with rrnB P1 is as rapid as, or more rapid than, that reported for any other E. coli or bacteriophage promoter.     

Separation of E. coli tRNA-Tyr precursor RNA from E. coli tRNA by hydroxyapatite chromatography

An assay procedure is described for triosephosphate isomerase based on measurement of the ellipticity of l-glyceraldehyde 3-phosphate remaining when d,l-glyceraldehyde 3-phosphate is the source of substrate and d-glyceraldehyde 3-phosphate is converted by triosephosphate isomerase to dihydroxyacetone phosphate. The assay method has advantages over the conventional coupled-enzyme assays in that it circumvents the difficulties posed by instability of the coupling enzymes and their cofactors, as well as by inhibitors of triosephosphate isomerase which may be present in preparations of the coupling enzymes. Although the method is not suited for routine assays during purification or in most clinical applications, it has advantages for detailed kinetic studies where pH, temperature, or other factors cause the coupled-enzyme assay procedures to be unreliable.     

Physico-chemical study of complex formation of DNA with wild-type and mutant E. coli RNA polymerases. Recognition properties of beta-subunit

Complex formation of T₇ DNA with RNA polymerase from E. coli B/r WU-36-10-11-12 (E. coli W 12) and its rifampicin-resistant mutant rpoB409 was studied. The rpoB409 mutant possesses a highly pleiotropic effect due to alteration in the RNA polymerase β-subunit structure. The two RNA polymerases have been previously shown to differ in gene selection during RNA synthesis on T₇ DNA. In this study it was found that the change in selective properties of the mutant RNA polymerase occurs during its interaction with DNA, the general ability of the enzyme to melt DNA being unaffected.     

Inclusion bodies in recombinant E. coli producing human calcitonin tetramer, as visualized by immuno-gold electron microscopy

Inclusion bodies are described in recombinant E. coli cells harboring plasmid for the expression of a synthetic gene coding for human calcitonin tetramer. The inclusion bodies are visualized by electron microscopy and the protein is identified by immuno-gold technique, using antibodies against synthetic human calcitonin. The diameter of the inclusion bodies is 1 micron on the average.