Binding of Escherichia coZi RNA Polymerase to T7 DNA DISPLACEMENT OF HOLOENZYME FROM PROMOTER COMPLEXES BY HEPARIN*

Escherichia coli RNA polymerase holoenzyme bound to promoter sites on T7 DNA is attacked and inactivated by the polyanion heparin. The highly stable RNA polymerase-T7 DNA complex formed at the major T7 A1 promoter can be completely inactivated by treatment with heparin, as shown by monitoring the loss of activity of such complexes, and by gel electrophoresis of the RNA products transcribed. The rate of this inactivation is much faster than the rate of dissociation of RNA polymerase from promoter complexes, and thus represents a direct attack of heparin on the polymerase molecule bound at promoter A1. Experiments employing the nitrocellulose filter binding technique suggest that heparin inactivates E. coli RNA polymerase when bound to T7 DNA by directly displacing the enzyme from the DNA. RNA polymerase bound at a minor T7 promoter (promoter C) is much less sensitive to heparin attack than enzyme bound at promoter A1. Thus, the rate of inactivation of RNA polymerase-T7 DNA complexes by heparin is dependent upon the structure of the promoter involved even though the inhibitor binds to a site on the enzyme molecule.


Escherichia
coli RNA polymerase holoenzyme bound to promoter sites on T7 DNA is attacked and inactivated by the polyanion heparin. The highly stable RNA polymerase T7 DNA complex formed at the major T7 Al promoter can be completely inactivated by treatment with heparin, as shown by monitoring the loss of activity of such complexes, and by gel electrophoresis of the RNA products transcribed. The rate of this inactivation is much faster than the rate of dissociation of RNA polymerase from promoter complexes, and thus represents a direct attack of heparin on the polymerase molecule bound at promoter Al. Experiments employing the nitrocellulose filter binding technique suggest that heparin inactivates E. coli RNA polymerase when bound to T7 DNA by directly displacing the enzyme from the DNA. RNA polymerase bound at a minor T7 promoter (promoter C) is much less sensitive to heparin attack than enzyme bound at promoter Al. Thus, the rate of inactivation of RNA polymerase .T7 DNA complexes by heparin is dependent upon the structure of the promoter involved even though the inhibitor binds to a site on the enzyme molecule.
Heparin has been shown to be an effective inhibitor of bacterial and eukaryotic DNA dependent RNA polymerases (l-3). Heparin is a highly sulfated linear polysaccharide (4). This polyanion inactivates free RNA polymerase at a faster rate than RNA polymerase which is bound to DNA (1). Sedimentation studies (1) and filter binding experiments (2) suggest that heparin inhibits RNA polymerase by forming a complex with the enzyme, possibly at the site involved in binding of the DNA template. The polyanionic character of the heparin molecule appears to be responsible for this binding; enzymes such as ribonuclease, which resemble RNA polymerase in their request for a substrate bearing regularly repeating negative charges, are also inhibited by heparin. In addition, a number of other polyanions are also inhibitors of RNA polymerases including polyethanesulfonate (51, aurintricarboxylic acid (61, and various polynucleotides such as poly(rI), poly(rU), and RNA (7-12).
Binary complexes of RNA polymerase bound to T7 DNA formed at low temperatures or elevated ionic strength are much more sensitive to attack by heparin than are the corresponding complexes formed at elevated temperatures and low ionic strength (2,12). For this reason many authors have used heparin to study the properties of promoter complexes (13-16).
In particular it is frequently assumed that "nonspecific" complexes formed by RNA polymerase at randomly encountered sites on the DNA template are attacked by heparin, while "specific" complexes formed at promoter sites are not. We were led to explore this matter in more detail by the observation that complexes formed by Escherichia coli RNA polymerase holoenzyme at T7 promoters could be rapidly inactivated by heparin (14,17).
When T7 DNA is employed as template, E. coli RNA polymerase catalyzes the synthesis of RNA which is predominantly of a single class size (2 to 2.4 x 10"; (18)) and which is initiated at three promoter sites (Al, A2, A31 clustered near the left end of the genome (19,20). When RNA polymerase is added to T7 DNA (in the absence of nucleoside triphosphates) at a concentration in excess of that needed to saturate these A promoter sites, it binds tightly at several additional promoter sites designated T7 promoters B, C, D, and E (17). These have been designated "minor" promoter sites since E. coli RNA polymerase preferentially uses the A sites when given a choice. Enzyme bound at these minor promoters forms highly stable complexes which closely resemble those formed at Al, A2, and A3 in terms of being able to initiate RNA chains rapidly (12,17). (These are "open" promoter complexes in terms of current models for transcription (131.) However, the rate of formation of complexes at these minor promoters is reduced as compared to the A promoter sites (17). The RNA products from all of these promoters can easily be separated and identified by gel electrophoresis, which provides a direct method for comparing the susceptibility of several T7 promoter complexes to inactivation by agents such as heparin. then pseudo-first order kinetics might be expected from the simplest reaction mechanism. In fact, the graph of In (fractional activity) as a function of time does not give a simple linear relationship, from which we conclude that the reaction is not a simple second order process. There may well be several steps involved in the displacement.
Alternatively we cannot rule out the possibility that there are several forms of heparin, perhaps varying in their density of negative charge, which differ significantly in their intrinsic rates of attack on RNA polymerase complexes.
Inactivation of RNA polymerase at Other T7 Promoters by Heparin -When RNA polymerase is allowed to bind to T7 AD111 DNA at a high enzyme to DNA ratio, the enzyme forms highly stable complexes at the T7 minor promoters C, D, and E in addition to promoter Al (17). When kinetic experiments carried out under these conditions are analyzed by gel electrophoresis one obtains a direct comparison of the sensitivity of complexes at these four T7 promoters to attack by heparin. Experiments were carried out at an enzyme to DNA ratio of 5, using the experimental protocol described above. Heparin was added to final concentrations of 0.01, 0.1, and 1.0 mg/ml and the ability of complexes to incorporate labeled nucleotides into RNA was assayed after increasing periods of heparin exposure (Fig. 1, right). The kinetic form of these reactions is different than that seen at left in Fig. 1. The curves reach a slowly decaying plateau, and the complexes require substantially higher concentrations of heparin for inactivation. We attribute these differences to the fact that RNA polymerase holoenzyme bound at promoter C is far more resistant to attack than that bound at promoter Al. This was suggested in the first experiment; however, under the conditions of Fig. 1, right, a substantial portion of the enzyme is bound at this promoter. This hypothesis was verified by electrophoretic analysis of the RNA products synthesized in this experiment (Fig. 2, right). At a heparin concentration of 0.2 mg/ml, species Al RNA completely disappears after complexes have been treated for 10 min (Track b), and species C RNA just begins to decrease in intensity at&r 90 min (Track d). As shown previously (171, RNA polymerase bound at promoter E is most sensitive to attack by heparin. Thus the inactivation of RNA polymerase*T7 DNA complexes by heparin varies considerably for these three different promoter sites and, interestingly, shows no direct correlation with the "strength" of the promoter. An experiment identical to Fig. 2, right, was also carried out using wild type T7 DNA at an enzyme concentration sufficient to saturate the three A promoter sites (data not shown). Under these conditions, enzyme bound at sites giving rise to species A RNA was attacked by heparin at a rate comparable to that found for AD111 in Fig. 2, left. We conclude that binary complexes formed at promoters A2 and A3 are at least as sensitive to heparin attack as promoter Al, although we have not measured the rate of attack at the individual A promoter sites.
Displacement of RNA Polymerase from Promoter Complexes by Heparin -We have shown above that heparin inactivates RNA polymerase when bound to T7 promoters in a highly stable complex. There are several plausible reaction mechanisms for this inactivation; one mechanism involves the physical displacement of the enzyme from the DNA. To test this possibility, nitrocellulose filter binding experiments were carried out according to the procedure of Hinkle and Chamberlin (12). This technique involves mixing RNA polymerase with a radioactively labeled DNA followed by filtration through a nitrocellulose filter. RNA polymerase . DNA complexes are retained on the filter (9, 12) and can be quantitatively determined by measurement of the radioactivity bound. If heparin displaces the enzyme from the complex, the DNA will not be retained, except for a low (2 to 3%) background level. The experiment was carried out as follows: complexes of RNA polymerase bound to T7 [3H]DNA were formed at an enzyme to DNA ratio of about 1.3, and heparin was added to a final  Fig. 3, top. The complex is disrupted with a half-time of about 27 min, while the half-time of inactivation for these complexes (Fig. 1, left) is also approximately 27 min. Thus, the rate of heparin displacement of enzyme from the promoter is approximately equal to the rate of complex inactivation.
Similar experiments were carried out at an enzyme to DNA ratio of about 5.2, at a heparin concentration of 1 mg/ml (Fig. 3,middle). This concentration of heparin inactivates all the complexes present under these conditions with a half-time of about 6 min ( Fig. 1, right). The corresponding half-time for disruption of the complex as measured by the nitrocellulose filter binding technique (see Fig. 3, middle) is about 7 min. Because the rate of heparin inactivation of enzyme. DNA complexes and the rate of heparin displacement of enzyme from the DNA are virtually identical, we conclude that heparin inactivates RNA polymerase T7 DNA complexes by directly displacing the enzyme from the DNA. It cannot be ruled out, of course, that heparin simply adds to the binary complex and that this ternary complex is not retained on the nitrocellulose filter. We consider this unlikely, since a very large number of protein.ligand complexes are retained on the filters, and we can see no reason why the presence of heparin bound to RNA polymerase should dramatically alter its ability to be retained. Under conditions where RNA polymerase is bound to several promoter sites per DNA molecule, one predicts that at an intermediate heparin concentration, some of the binary complexes will not be disrupted. For example, complexes formed at promoter C should not be disrupted after short periods of exposure, whereas complexes at promoter Al should be displaced (see above). This prediction was tested in the following manner: RNA polymerase was mixed with T7 PHIDNA at an enzyme to DNA ratio of about 5.2, as described above. The disruption of complexes was examined by nitrocellulose filter binding at a heparin concentration of 0.01 mglml (Fig. 3,  bottom). As expected, we observed a slight decrease in 13H]DNA bound to the filter, which presumably represents the decrease in efficiency of binding due to the displacement of some but not all of the enzyme molecules bound. However, the major fraction of DNA remains nonfilterable; that is, the more resistant complexes are stable to disruption by heparin, and these complexes do not lose their binding capacity.

DISCUSSION
Heparin is useful as a selective inhibitor of free RNA polymerase and of certain nonspecific RNA polymerase . DNA complexes (1,2,15,16). While it was previously believed that highly stable, open promoter complexes were resistant to attack by this inhibitor, we show here that complexes formed at all of the T7 promoters we have studied are attacked. However, the rate of attack on RNA polymerase bound at different promoter sites can vary over a considerable range. Surprisingly, the complexes formed at the major T7 A promoter site are considerably less stable than those formed at the minor promoter C. Under conditions of free site selection most E. coli RNA polymerase holoenzyme binds at the A promoter site. Thus heparin is not a reagent with which "strong" promoter compIexes can be selectiveIy isolated, unless one has previous knowledge of the heparin sensitivities of the promoters involved.
Several criteria were used to establish that the inhibitory activity of our heparin preparations was not due to a minor contaminant.
First, our preparations contained no detectable RNase or DNase. Second, similar levels of inhibition were observed with heparin from several sources, and with heparin which had been fractionated by exclusion gel chromatography. Finally, spermidine, which would presumably interact with this polyanion, reduced the amount of inhibition substantially when present in the reaction mixtures (data not shown).
Why do different binary complexes differ in their sensitivity to heparin? One possibility we have considered is that different promoter sites have different affinities for RNA polymerase: E + promoter + E -promoter.
Since RNA polymerase binds heparin tightly: E + heparin e E:heparin then at equilibrium in the presence of an appropriate concentration of heparin, the inhibitor would selectively remove RNA polymerase from promoters with the lowest affinity for the enzyme. This would not be inconsistent with the order of heparin sensitivity we observe: we have shown elsewhere (12) that the major A promoters do not bind RNA polymerase more strongly than C and E; the selection of A as a major promoter appears to be due to differences in the rate of stable complex formation (17).
While this is a plausible explanation, it seems to us less likely than the notion that RNA polymerase bound at different promoters is attacked at different rates by heparin because the enzyme differs in its conformation in those different complexes. The time required to reach equilibrium by the first model is large, since the rates of dissociation of RNA polymerase from DNA and from heparin are very slow. We have tried but have not been able to observe a true equilibrium in experiments such as those of Fig. 1; enzyme. heparin complexes do not dissociate at a significant rate in our hands even in the presence of excess DNA. In addition to these quantitative arguments, we have shown elsewhere that RNA polymerase bound at these different promoter sites is also attacked at different rates by the drug rifampicin (171, which binds at a different site on the RNA polymerase molecule (35) from that used by heparin. Taken together all of these observations favor a model in which RNA polymerase conformation is altered by the structure of different promoter sites; these different conformations are attacked at quite different rates by inhibitors of the enzyme. This suggests the definite possibility that these different conformations might also respond selectively to different regulatory molecules.