Spatial relation of the σ subunit and the rifampicin binding site in RNA polymerase of Escherichia coli

Abstract

sigma subunit of Escherichia coli RNA polymerase is known to stimulate specific RNA chain initiation. Rifampicin, an inhibitor of RNA chain initiation, binds to a single site on the beta subunit of RNA polymerase. We have used the fluorescence energy transfer technique to deduce proximity relationships of sigma subunit and rifampicin binding site on the enzyme. Isolated sigma subunit was covalently labeled with fluorescent donors in two ways: specific labeling of a single sulfhydryl residue with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate (1,5-I-AENS) and nonspecific labeling on the surface of the protein with dansyl chloride (Dns-Cl) adsorbed on Celite. The labeled sigma subunits were biologically active and formed a stoichiometric complex with core polymerase. The efficiency of energy transfer was obtained from the fluorescence intensity and the excited-state lifetime of the sigma-labeled holoenzyme in the presence and absence of rifampicin, which served as an energy acceptor. The transfer efficiency (2%) from AENS to rifampicin placed AENS somewhere between 42 and 85 A away from the rifampicin binding site. The rotational mobility of the donor was determined by nanosecond fluorescence depolarization spectroscopy, while the acceptor orientation was assumed to be fixed at some unknown angle. The efficiency measured for energy transfer from Dns to rifampicin was 10% in the presence of 0.2 M KCl. The distance from the surface of sigma subunit to the rifampicin binding site was calculated to be 27--38 A for a model having a randomly distributed and oriented array of donors on the surface of a spherical sigma subunit of 31-A radius. Our results indicate that rifampicin does not inhibit the initiation of transcription by RNA polymerase through a direct interaction with sigma subunit. In addition, energy transfer measurements under low salt conditions suggest that in RNA polymerase dimer the two rifampicin binding sites are symmetric with respect to each sigma subunit.

Determination of a Binding Site for a Non‐Substrate Ligand in Mammalian Cytosolic Glutathione S‐Transferases by means of Fluorescence‐Resonance Energy Transfer

“…FRET distance ( R ) values were calculated to be 2.21 nm and 1.82 nm for the class-a and class-n GST, respectively. FRET has been used successfully to calculate molecular distances in other multisubunit enzymes and assemblies (Wu and Wu, 1974;Wu et al, 1976;Sheperd and Hammes, 1976 (---), 1.82-nm and 2.21-nm arcs formed by the FRET-determined radii for the class-x and class-a isoenzymes, respectively. Tryptophan residues (W) in class-n and class-a GST are indicated in bold.…”

The orientational freedom of molecular probes. The orientation factor in intramolecular energy transfer

“…In the 30 years or so since Frster developed an exact quantum mechanical theory of resonance energy transfer for the "very weak" dipole-dipole coupling limit (Frster, 1948(Frster, , 1951(Frster, , 1965, an extensive and still rapidly growing literature has described the utilization of the phenomenon to determine intramolecular separations (e.g., recently Wu et al, 1976;Langlois et al, 1976;Shepherd et al, 1976;Wright and Takahashi, 1977;Papadakis and Hammes, 1977;Zukin et al, 1977) and conformational dynamics (Haas et al, 1975(Haas et al, , 1977Ohmine et al, 1977) of mainly biological macromolecules. Further impetus to such studies was undoubtedly provided by the finding that the predicted inverse sixth-power distance dependence of the transfer rate holds closely for several series of oligomers having donor (D) and acceptor (A) moieties bound covalently at their ends, the separation of which depends on the number of intermediate monomer units (Stryer and Haugland, 1967;Conrad and Brand, 1968;Gabor, 1968).…”

Interaction of an Antituberculosis Drug with Nano‐sized Cationic Micelle: Förster Resonance Energy Transfer from Dansyl to Rifampicin in the Microenvironment

“…We have determined the values of the parameters m, k q and k 0 by fitting the decay curves of the donor (DC) in the excited state in the absence and presence of increasing concentration of quencher molecule (RF) to Eq. (14). Figure 3b shows the time resolved fluorescence transients of DC bound to CTAB micelle in absence and presence of RF molecules, fitted with Eq.…”

“…Figure 3b shows the time resolved fluorescence transients of DC bound to CTAB micelle in absence and presence of RF molecules, fitted with Eq. (14). The observed fluorescence transients were fitted using a nonlinear least squares fitting procedure (software SCIEN-TIST TM ) to a function ðXðtÞ ¼ R t 0 Eðt 0 ÞPðt À t 0 Þdt 0 Þ comprising of the convolution of the IRF (E(t)) with exponential [P*(t) = P*(0) exp{)k 0 t ) m[1 ) exp()k q t)]}].…”