Rotational diffusion of Escherichia coli RNA polymerase free and bound to deoxyribonucleic acid in nonspecific complexes.
We have studied the rotational diffusion of Escherichia coli RNA polymerase free in solution and bound nonspecifically to DNA fragments. The rotational motion was measured by the decay in anisotropy of the triplet-triplet absorption by using as probes either the liganded enzyme inhibitor Rose Bengal or eosin 5'-isothiocyanate conjugated to the protein. The time resolution extended from 10 ns to 1 ms. Free RNA polymerase (holoenzyme) at high salt concentration (1 M NaCl) is monomeric and diffuses at 5 degrees C with a rotational correlation time of 0.66 microseconds, corresponding to an equivalent hydrodynamic sphere with a radius of 7.4 nm. These values and the known molecular weight are most compatible with a nonspherical shape, e.g., an oblate ellipsoid with an axial ratio of about 3. In 0.1 M NaCl, the holoenzyme is dimeric and has a rotational correlation time of 2 microseconds. The decay of anisotropy is at least biexponential upon binding RNA polymerase to calf thymus DNA or to poly[d(A-T)]. The fast component with half of the amplitude has decay kinetics comparable to those seen with the free monomeric enzyme. The slow component has a rotational correlation time of about 14 microseconds and is independent of DNA chain length in the range greater than 180 base pairs. Both rotational correlation times decrease with temperature, and the relative amplitudes change such that the faster component dominates at higher temperature. The rotational relaxation of the enzyme-DNA complexes is discussed in terms of alternative models involving rigid rod-sphere diffusion, conformational changes in the enzyme and/or DNA, sliding motions of the protein along the DNA, and torsional-bending motions of DNA envisioned as a deformable rod.
D002942:Circular Dichroism; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D004058:Diffusion; D004926:Escherichia coli; D009038:Motion; D009876:Operon; D011067:Poly dA-dT; D011485:Protein Binding; D013696:Temperature
D011067:Poly dA-dT; D004247:DNA; D012321:DNA-Directed RNA Polymerases