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F Y Wu

Publications and source records attributed to F Y Wu.

At least 91 records · Page 5Linked to original sources
PubMed1976

Conformational transition of Escherichia coli RNA polymerase induced by the interaction of sigma subunit with core enzyme.

The isolated sigma subunit of Escherichia coli RNA polymerase has been labeled covalently with a fluorescent probe, N-(1-pyrene)maleimide. The labeled sigma subunit (PM-sigma) still retained its biological activity in stimulating transcription of T7 DNA by core enzyme. When a stoichiometric amount of core enzyme was added to a solution of PM-sigma, there was a decrease in fluorescence intensity without shifts in emission maxima of PM-sigma. The kinetics of the interaction between the sigma subunit and core enzyme was investigated with the stopped-flow technique by monitoring the fluorescence quenching. A biphasic change of fluorescence intensity with respect to time was observed when PM-sigma was rapidly mixed with an excess of core enzyme. The kinetic data can be analyzed in terms of a mechanism in which a fast bimolecular binding of sigma to core enzyme is followed by a relatively slow isomerization of the holoenzyme formed. From the best-fit kinetic parameters, an overall binding constant of less than or equal to 3X10(-10)M was estimated for the PM-sigma core complex, in agreement with that obtained by the fluorimetric titration. In addition, we have studied the effect of temperature on the rate constant associated with the conformational change of the holoenzyme, which shows a temperature transition around 20 degrees C. The nonlinear Arrhenius plot obtained implies that the conformational transition is complex and may be composed of several processes. The activation energy for the "overall" conformational change was estimated to be 6.7 kcal/mol. The kinetic evidence for the conformational transition of holoenzyme induced by the interactions of sigma subunit with core enzyme presented here further supports the proposition that the sigma subunit acts on core enzyme to trap a unique conformation of RNA polymerase which recognizes the proper promoters and initiates the synthesis of specific RNA chains.

DNA-Directed RNA Polymerases↗
PubMed1976

Molecular mechanism of the rifampicin -RNA polymerase interaction.

Equilibrium and kinetic studies of the interaction of rifampicin with RNA polymerase of Escherichia coli were performed by exploiting the quenching of intrinsic fluorescence of the protein by the drug. Fluorimetric titrations show that rifampicin binds stoichiometrically to the core and holoenzyme with an apparent Kd of less than or equal to 3 x 10(-9) M. Neither the addition of template nor the formation of the initiation complex in the presence of dinucleotide and nucleoside triphosphate prevents the rifampicin-enzyme interaction. Although the equilibrium binding constant for the rifampicin-RNA polymerase complex is about the same for the core and holoenzyme and the holoenzyme-T7 DNA complex, stopped-flow studies indicate that the rates at which rifampicin interacts with these enzyme forms are different. In all three cases, the kinetic data can be interpreted in terms of a mechanism in which the rapid bimolecular binding of rifampicin to RNA polymerase is followed by a relatively slow isomerization of the drug enzyme complex: (See article). While the values of dissociation constant K1 = (k-1/k1), for the first binary complex (ER) are similar, the rate constant for the forward isomerization, k2, decrease in the order of core enzyme greater than holoenzyme greater than the holoenzyme-T7 DNA complex. The fact that this order is parallel to the relative rates of inactivation of the enzymes and the enzyme-DNA complex suggests that the inactivation may be due to the rifampicin-induced isomerization (conformational change) of the enzyme. This is supported by our observations that an enzyme complex which is in the process of elongating RNA chains can still bind rifampicin, although the enzyme activity is not inhibited by such binding. The values of overall binding constants calculated from the kinetic parameters, 1-2 x 10(-9) M, are in good agreement with the values of the apparent Kd obtained from fluorimetric titrations and Ki determined by enzymatic assays. In addition, the observations that the formation of an initiation complex leads to a significant but not complete rifampicin-resistant RNA synthesis and the recent finding that rifampicin only partly inhibits the formation of the first phosphodiester bond in an abortive initiation of RNA chains are consistent with our kinetic mechansim, i.e., the existence of two forms of the rifampicin-RNA polymerase complex, only one of which is able to initiate the RNA chains.

Binding Sites↗
PubMed1976

Spatial relationship of the sigma subunit and the rifampicin binding site in RNA polymerase of Escherichia coli.

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.

Binding Sites↗
PubMed1975

Sigma cycle during in vitro transcription: demonstration by nanosecond fluorescence depolarization spectroscopy.

Studies of RNA chain initiation have suggested that the sigma subunit of Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate: RNA nucleotidyltransferase; EC 2.7.7.6) is released from the enzyme-template complex during transcription and may be reused by another core polymerase. Nanosecond fluorescence depolarization spectroscopy was used to follow the sigma cycle. Isolated sigma subunit labeled with the fluorescent probe dansyl (DNS) chloride bound stoichiometrically to core polymerase and stimulated transcription of phage T7 DNA to the same extent as did unlabeled sigma. DNS-sigma showed an exponential fluorescence anisotropy decay corresponding to a rotational correlation time of about 100 nsec. This value was unaffected by addition of T7 DNA, but increased about 6-fold when core polymerase was added, and increased further when T7 DNA was added. Such increases are expected for the formation of molecular complexes. Using the anisotropy decays for free DNS-sigma and DNS-sigma-core enzyme bound to T7 DNA, we calculated theoretical decay curves for various mixtures of free and bound sigma. Comparison of the observed anisotropy decay with the calculated curves indicated that about 55% of DNA-sigma was released from the enzyme-T7 DNA complex in the presence of four nucleoside triphosphates under low salt conditions. Sigma release did not occur if rifampicin was added prior to addition of four nucleoside triphosphates or if only three nucleoside triphosphates were present. After sigma was released, addition of core polymerase with rifampicin reduced the free sigma to less than 15%, indicating that the released sigma was accessible to the added core enzyme. Thus these studies have provided physical evidence for the sigma cycle during in vitro transcription.

Binding Sites↗