Molecular mechanism of the rifampicin-RNA polymerase interaction

Abstract

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.

Evidence that rifampicin can stimulate readthrough of transcriptional terminators inEscherichia coli,including the attenuator of therpoBCoperon

“…What could be the mechanism of such a general effect? There is evidence that rifampicin can bind stoichiometrically to RNA polymerase which is actively transcribing, without inhibiting elongation (11). Thus it seems possible that the drug could bind to enzyme molecules after they have initiated transcription in our in vivo conditions (either during elongation, or when the polymerase has paused at a terminator), and could subsequently interfere directly in some general step of termination (reviewed in 42).…”

“…There is evidence, however, that certain growth constraints can partially uncouple the expression of the rpo and rpl genes in the above operon (reviewed in 1,2,8,9). One such constraint is produced by the drug rifampicin, which binds to RNA polymerase and blocks initiation (but not elongation) of the RNA product (10,11 116T 229 T-T15 335 7ST69). absolute increase in the rate of synthesis of S and W' (and other polymerase subunits) without a parallel effect on the ribosomal proteins L7/12 encoded upstream in the 55' operon (12)(13)(14)(15)(16).…”

Rifampin-induced initiation of chromosome replication in dnaR-deficient Escherichia coli cells

“…Effects of recA and dnaA mutations on Rif-induced DNA synthesis. DNA synthesis in strains HC5127 ⌬recA306 dnaR130, HC5133 dnaA167 dnaR130, HC5112 dnaA167 dnaR ϩ , and pSA96 dnaA ϩ plasmid-carrying HC5133 was measured at 30ЊC (--⅜--) and 42.5ЊC (--ⅷ--), and with addition of 10 g of Rif per ml just before (--⅜--) or 90 min after (--ⅷ--) the upshift, as described in the legend to Fig. 1.…”

“…HC5103 cells were grown at 30ЊC in LP broth containing [ 14 C]thymine at 0.1 Ci/ml from a cell density of about 5 ϫ 10 6 /ml to a cell density of about 8 ϫ 10 7 /ml. The culture was incubated at 30ЊC (--⅜--) and 42.5ЊC (--ⅷ--), and with addition of Rif to a concentration of 3 (--Ç--), 10 (--⅜--), 30 (--É--), or 100 g/ml at the time of the upshift (--Ⅺ--) (A). To another portion of the culture, Rif and Cam were simultaneously added to concentrations of 10 and 200 g/ml, respectively, at the time of the upshift (--f--) (B).…”

“…DNA synthesis in strain HC5103 with the rpoB60 mutation was measured at 30ЊC (--⅜--) and 42.5ЊC (--ⅷ--) and with addition of 10 g of Rif per ml at the time of the upshift (--⅜--), as described in the legend to Fig. 1.…”