A model for the quaternary structure of Escherichiu coli RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) is presented. It is based on results from classification of profiles of enzyme molecules, and from application of immuno electron microscopy.Classification of molecules, prepared with the single carbon layer technique, was first achieved for images recorded in dark field with the scanning transmission electron microscope and later on for images recorded in brightfield transmission electron microscopy. It results in five approximately equally sized groups, containing about 80 "/, of the core enzyme profiles. Holoenzyme profiles can be grouped into the same classes, and have approximately the same dimensions (9nm x 16nm). Based on the shapes and sizes of the classified profiles, a tentative model for core enzyme has been constructed.Correlation of shadow projections of this model, with the distributions of attachment sites of antibodies against CI,/I$' and CT over thc profiles, has led to models for core and holoenzyme in which the subunits are localized.The model is compared with literature data on the quaternary structure of RNA polymerase.Eschuiclziu coli RNA polymerase (nucleosidetriphosphate : RNA nucleotidyl transferase) catalyses RNA synthesis. Functional aspects of the enzyme have been extensively studied; for reviews, see 18, 91. Various biochemical and physical techniques have been used to study the quaternary structure of RNA polymerase; for a review, see [lo].In cross-linking studies, /I-/? was found to be the main product for both core and holoenzyme [ll, 121. Using crosslinkers of different lengths, Hillel and Wu [12] found distances of less than 1.2nm between subunits. They presented a model consisting of spherical subunits in which / I and p' form a tetrahedron-like structure together with the M subunits, which constitute a dimer; in holoenzyme the CT subunit interacts with fl and /Y, and with one CI subunit. Partial proteolysis studies [13 -151 support this model. Stender [I61 compared the accessibility of free subunits with their accessibility in situ, using antibodies raised against isolated subunits. He found the CI subunit in situ to be much less accessible than in the isolated state; for b and /j' this difference is much smaller.On the basis of small-angle neutron scattering data, Stockel et al. [I71 presented a model in which the core enzyme is an extended triangle of elongated C I~, /I, and /Y subunits, of which the latter two are curved. They arrived at an axial ratio of 6.1Abbreviurions. TEM, transmission electron microscope/y ; STEM, Enzyme. Escherichia coli RNA polymerase (EC 2.7.7.6).scanning transmission electron microscope/y. for C I~ and /Y, and of 5.6 for 1. The distances between the centres of mass of these subunits range from 7.0 nm to 8.2 nm. In the holoenzyme the CT subunit, for which they derived the shape of an elongated disc, nestles up to the core enzyme in a spacefilling manner; the centre-of-subunit structure forms a tetrahedron 1181.Us...

In the case of RNA polymerase holoenzyme the radioactive substituents were evenly distributed between subunits p and 0. Apparently the topology of the rifamycin binding site of holoenzyme, similarly to core enzyme, precludes attacks of nucleophilic functions from p' and a, but it allows nucleophilic functions from subunits p and 0 to react with equal probability on BrAcNEtS-Rif. In the presence of a 20-fold excess of pOH-HgBzSO,H, the modification of holoenzyme was drastically altered. Virtually all substitution took place on subunit p', very little on / 3 and none on subunits 0 and a.The antibiotic rifamycin and its various derivatives inhibit bacterial RNA synthesis [I] in vitro and in vivo by binding to RNA polymerase [2,3]. It was shown that RNA synthesis becomes resistant to inhibition by the drug once chain initiation has been accomplished [4,5]. Rifamycin derivatives do not prevent the attachment of DNA templates to RNA polymerase [6,7] but seem to interfere with the binding of purine triphosphates [8], thus preventing initiation, e.g. formation of the first phosphodiester bond. Uncertainty exists with respect to the detailed mode of action of rifamycin. Does rifamycin inhibit by direct competition with ATP or GTP at the initiation site or in an allosteric manner, after binding to a site distinct from the initiation site [9]? Genetic experiments provide evidence that the drug binding site resides in subunit p [lo]. The isolated subunit p, however, did not show a perceptible affinity for rifamycin [ll], indicating that the drug binding site is generated on subunit j after association with other subunits. Affinity labelling studies with substrate and template analogues were recently employed successfully to explore the topology of the respective binding sites. Evidence was obtained that the bound substrate is in contact with subunits j and B' [12,13], whereas the template binding site is located at subunit p' [12].It appeared to us that an inhibitor like rifamycin which is relatively easy to derivative and has a high affinity towards the enzyme would be very suitable for the purpose of selective modification. This paper describes the modification of RNA polymerase from Escherichia coli with a 14C-labelled alkylating derivative of rifamycin SV.Abbreviations. BrAcNEtS-Rif, 3-(2-bromoacetamidoethyl)-thio-rifamycin SV; pOH-HgBzOH, p-hydroxybenzoic acid; pOHHgBzSO,H, p-hydroxymercuribenzene sulfonic acid; [poly(dT)] . [r(Ap),A], hybrid formed between poly(dT) and r(Ap),A. Other abbreviations for nucleotides and polynucleotides follow CBN rules, see Eur. J. Biochem. 15, 203 (1970).Enzyme. DNA-Dependent RNA polymerase (EC 2.7.7.6). MATERIALS AND METHODS EnzymesRNA polymerase from E. coli was purified according to Zillig et al. (spec. act. 75 nmol ATP x min-' x mg protein-' with calf thymus DNA as template) Eur. J. Biochem. 56 (1975)

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The synthesis of dimeric derivatives of rifamycin SV differing in the length of spacer, of derivatives of rifamycin SV possessing 2,4-dinitrophenyl groups in varying distances relative to the aromatic part of the antibiotic and a derivative of rifamycin SV carrying a biotinyl residue is described. Rifamycin SV covalently attached to bovine serum albumin was employed to produce antibodies against rifamycin SV in rabbits. Rifamycin SV as well as 2,4-dinitrophenyl-specific antibodies and avidin were used to study the interaction of RNA polymerase with the respective derivatives of rifamycin SV. The results of this investigation can be summarized as follows.1. Antibodies, which are specific for rifamycin SV do not recognize the rifamycin SV molecule in the enzyme . antibiotic complex.2. 2,4-Dinitrophenyl-specific antibodies are unable to recognize the 2,4-dinitrophenyl residue in the complex enzyme . 2,4-dinitrophenylaminoethylthio-rifamycin-SV.3. However, 2,4-dinitrophenyl groups in complexes between enzyme and 2,4-dinitrophenyl derivatives of rifamycin SV are recognized, if those are separated far enough from the rifamycin part.4. RNA polymerase binds to the rifamycin SV portion in complexes avidin . biotinylaminoethylthio-rifamycin-SV.5. Dimeric rifamycin SV molecules do not form ternary complexes with RNA polymerase. From those results it is concluded that the binding site of RNA polymerase for rifamycin SV extends 1.40 -1.90 nm deep into the interior of the enzyme structure and that the ansa chain of the antibiotic extends furthest into the enzyme Rifamycin SV and its derivaties are strong inhibitors of DNA-dependent RNA synthesis in bacteria [I -51. They interfere with chain initiation of RNA synthesis when binding to the enzyme, RNA polymerase, or to the complex RNA-polymerase . DNA [6,7]. Recent investigations yield evidence that the first phosphodiester bond is still formed in the presence of the antibiotic and that the steps of the enzymatic reaction, which lead to the elongation of the phosphodiester bond, are inhibited [8]. Kinetic and thermodynamic studies reveal the high affinity of rifampicin and other rifamycin SV derivatives to enzyme or enzyme . DNA complexes [9-121. Little is known about the topology of the antibiotic binding site of Abbreviations. The abbreviations of the various synthetic derivatives of rifamycin SV are given in Table 2; Nzph = dinitrophenyl.Enzyme. DNA-dependent RNA polymerase (EC 2.7.7.6).RNA polymerase. An investigation of a rifampicininsensitive RNA polymerase from Escherichia coli and affinity labelling studies with chemically reactivederivatives of rifamycin SV demonstrate that the p subunit seems to carry the antibiotic binding site [13,14]. The affinity labelling experiments indicate that the bound rifamycin SV is in contact with o subunit. Structure-activity relations based on chemical modifications of the antibiotic affecting the activity indicate that both the ansa ring, as well as the aromatic part of the antibiotic, are involved in the interaction with the enzyme (F...

The interaction of sigma subunit of E. coli RNA polymerase with DNA, either double or single-stranded, and with two inhibitors of RNA synthesis was investigated by using antibodies directed against the subunit. Free sigma subunit was shown to interact with poly(dA), poly(dT), poly(dAC).poly(dGT), T7 DNA and, to a lesser degree, with lambda DNA. When the sigma subunit forms part of the holo enzyme, sigma also interacts with poly(dG).poly(dC). Rifampicin and streptolydigin interact with sigma in the holo enzyme and with free and core bound sigma subunit, respectively. The results suggest that sigma recognizes mainly AC-GT-sequences in double-stranded DNA. The findings are correlated with the base composition in RNA polymerase binding regions of promoters and suggest at least a general interaction between sigma subunit and single-stranded DNA in open complexes.