Inhibition of Azotobacter vinelandii RNA polymerase by cibacron blue F3GA.

Cibacron blue F3GA is a potent inhibitor of the Azotobacter vinelandii DNA-directed RNA polymerase. Addition of 8 micrometer Cibacron blue F3GA prior to initiation results in a greater than 90% inhibition of the poly[d(A-T]-directed synthesis of poly[r(A-U)] while addition of the dye during the course of the reaction is without effect on chain elongation. Binding of RNA polymerase to [3H]poly[d(A-T)] is inhibited by only 15% in the presence of 8 micrometer Cibacron blue F3GA. Inhibition by Cibacron blue F3GA is noncompetitive with regard to ATP, UTP, or template. The poly[d(A-T)]-directed pyrophosphate exchange reaction is relatively resistant to inhibition by Cibacron blue F3GA. Rifampicin added to a similar reaction (in the presence of absence of Cibacron blue F3GA) results in 95% inhibition of the exchange reaction. The interaction of the RNA polymerase core enzyme with Cibacron blue F3GA is shown by the formation of a difference spectrum with a positive maximum at 675 nm which is not affected by the presence of a high concentration (4 micrometer) of rafampicin. The data indicate that Cibacron blue F3GA acts by binding to RNA polymerase and inhibits a step between the synthesis of the initial phosphodiester bond and formation of a stable ternary elongation complex.

while addition of the dye during the course of the reaction is without effect on chain elongation.

Binding of RNA polymerase to ["H]poly[d(A-T)]
is inhibited by only 15% in the presence of 8 FM Cibacron blue F3GA. Inhibition by Cibacron blue FSGA is noncompetitive with regard to ATP, UTP, or template. The poly[d(A-T)l-directed pyrophosphate exchange reaction is relatively resistant to inhibition by Cibacron blue FSGA. Rifampicin added to a similar reaction (in the presence or absence of Cibacron blue F3GA) results in a 95% inhibition of the exchange reaction. The interaction of the RNA polymerase core enzyme with Cibacron blue F3GA is shown by the formation of a difference spectrum with a positive maximum at 675 nm which is not affected by the presence of a high concentration (4 FM) of rifampicin.
The data indicate that Cibacron blue F3GA acts by binding to RNA polymerase and inhibits a step between the synthesis of the initial phosphodiester bond and formation of a stable ternary elongation complex.
Cibacron blue FJGA, a sulfonated polyaromatic dye, has been shown to bind to several enzymes which interact with nucleotide substrates or nucleotide coenzyme ligands (l-3). Thompson et al. (1) and Thompson and Stellwagen (2) have proposed that the dye binds to the dinucleotide fold present in these proteins. DNA-dependent RNA polymerase can be envisaged to contain one or more such sites because of the number of nucleotide binding sites present on the enzyme such as the template binding region (41, product binding site (51, and initiation and elongation nucleotide binding sites (6-8). The present report describes the effects of Cibacron blue F3GA on RNA polymerase from Azotobacter uinelandii.
The results to be presented indicate that Cibacron blue inhibits RNA polymerase at a step subsequent to the synthesis of the initial phosphodiester bond and before the formation of a stable elongation complex.  The reactions contained (final volume 0.25 ml): 80 mM Tris/HCl. pH 7.8, 4 mM dithiothreitcl, 4 mM MgCL, 2.5 pg ofRNA polymerase holoenzyme, and the indicated concentration of Cibacron blue F3GA. After a preliminary incubation of 5 min at 37" the following were added to A: 9 nmol of polyld(A-T)], 0.4 mM UTP, 1 mM sodium 13*Plpyrophosphate and ATP, 5'-AMP, UpA, and rifampicin at the concentrations indicated above. Added to B: 9 nmol of polyld(I-01, 0.4 mM CTP, 1 mM sodium 132Plpyrophosphate and GTP at the concentrations indicated above. The incubation was for 10 min at 37". change reaction was also less inhibited by Cibacron blue than was the synthesis of poly[r(G-C)].

MATERIALS
In the presence of 16 PM Cibacron blue, the synthesis of polylr(A-II)] or polylr(G-01 would be almost completely inhibited. As shown in Table III, even at the high inhibitor concentration considerable pyrophosphate exchange activity remained. Both Cibacron blue and rifampicin (13) inhibit RNA polymerase when added prior to chain initiation and the ternary elongation complex is not affected by either inhibitor.
As indicated in Table III, the pyrophosphate exchange reaction is relatively insensitive to Cibacron blue but is markedly, but not completely, inhibited by rifampicin.
The data indicate that Cibacron blue is a noncompetitive inhibitor which acts by binding to a site (or sites) on the RNA polymerase core unit. Thompson and Stellwagen (2) have shown that the absorption spectrum of Cibacron blue bound to protein undergoes a red shift. As shown in Fig. 5, addition of Cibacron blue to RNA polymerase produced difference spectra with positive absorption maxima at 675 nm and an isosbestic point at 585 run. The increase in the absorbance at 675 mn exhibits a hyperbolic dependence on the concentration of the added dye (Fig. 5) indicating the formation of a saturated Cibacron blue. polymerase complex.
Using a saturating concentration of rifampicin (20 I*M) to form the rifampicin . polymerase complex, the subsequent addition of 8 PM Cibacron blue still produced the characteristic difference spectrum with a maximum at 675 nm (results not shown). This and the data presented in Table III indicate that each ligand binds to a separate site on the core enzyme.

DISCUSSION
The nature of the inhibition of RNA polymerase produced by Cibacron blue is in several respects similar to that of rifampitin. Inhibition is noncompetitive with respect to template and substrates (Fig. 2, A, B , and Cl and RNA polymerase engaged in the ternary elongation complex is not inhibited by Cibacron blue (Fig. 3)   least the first phosphodiester bond is not blocked by the inhibitor. Johnston and McClure (14) have shown that the initial dinucleoside tetraphosphate is still synthesized by promoteror polyld(A-Tll-bound RNA polymerase in the presence of rifampicin. While we have not as yet determined the nature of the product synthesized in the presence of high concentrations of Cibacron blue, the ability of the "inhibited" enzyme to catalyze the pyrophosphate exchange reaction is evidence for the formation of at least the first phosphodiester bond. That the mechanisms by which rifampicin and Cibacron blue inhibit RNA polymerase differ in detail is indicated by the marked inhibition of pyrophosphate exchange by rifampicin. We have carried out Cibacron blue challenge experiments analogous to the rifampicin challenge procedure of Mange1 and Chamberlin (15) using the polyld(A-T)]-directed reaction and found that inhibition was instantaneous,' suggesting that a different substep in the initiation reaction leading to the resistant elongation complex was affected by Cibacron blue. The noncompetitive nature of the inhibition by the dye and the demonstration that the synthesis and pyrophosphorolysis of at least the initial dinucleoside tetraphosphate can occur in the presence of Cibacron blue suggest that the sensitive step may be translocation. This is also the mechanism suggested for inhibition of RNA polymerase by rifampicin (14) and it is possible that Cibacron blue may prevent continued chain elongation by occluding the RNA product site. We are currently characterizing the products synthesized in the presence of Cibacron blue to determine whether a discrete size class is formed i.e. dinucleoside tetraphosphate or longer. Thompson and Stellwagen (2) have proposed that the forma-Znhibition of RNA Polymerase by Cibacron Blue tion of a difference spectrum having a positive maximum in the range 660 to 680 nm appears to be characteristic for complexation of Cibacron blue with proteins containing a supersecondary structure termed the dinucleotide fold (16, 17). The RNA polymerase core enzyme. dye complex produces a difference spectrum having a positive maximum at 675 nm. The data are suggestive of the presence of a dinucleotide fold structure in RNA polymerase and at least indicate the presence of a hydrophobic pocket which the dye occupies in order to produce a perturbation in the absorption spectrum of the dye chromophore. Acknowledgment-We thank Mr. Gopalan Nair for expert technical assistance. Halling et al. (18) have found that one of the subunits of urea dissociated Bacillus subtilis RNA polymerase is retained on a blue dextran/Sepharose column. Reconstitution experiments using subunits prepared from rifampicin-sensitive andresistant enzyme have shown that the subunit retained on the blue dextran/Sepharose column is the p' subunit.' Cibacron blue FJGA is the dye covalently attached to blue dextran (1). This suggests that Cibacron blue F3GA probably interacts with the p' subunit of the Azotobacter vinelandii RNA polymerase to produce the difference spectra and the inhibitory properties presented in this study.
Several dyes have now been shown to inhibit RNA polymerase by directly complexing with the enzyme. Congo red (19) and gallin (20) block template binding. Cibacron blue inhibits at a step subsequent to synthesis of the initial dinucleoside tetraphosphate and before formation of a ternary elongation complex. Rose bengal has been shown to be an inhibitor of chain elongation (21). It appears probable that other dyes will be found to inhibit this enzyme and may allow for a further dissection of the substeps involved in the RNA polymerase reaction.
Since the completion of this study we have found that the wheat germ RNA polymerase II (22) is inhibited by Cibacron blue F3GA and binds to Cibacron blue/Sepharose."