Active site labeling of the RNA polymerases A, B, and C from yeast.

RNA polymerases A, B, and C from yeast were modified by reaction with 4-formylphenyl-gamma-ester of ATP as priming nucleotide followed by reduction with NaBH4. Upon phosphodiester bond formation with [alpha-32P]UTP, only the second largest subunit, A135, B150, or C128, was labeled in a template-dependent reaction. This indicates that these polypeptide chains are functionally homologous. The product covalently bound to B150 subunit was found to consist of a mixture of ApU and a trinucleotide. Enzyme labeling exhibited the characteristic alpha-amanitin sensitivity reported for A and B RNA polymerases. Labeling of both large subunits of enzyme A and B but not of any of the smaller subunits was observed when the reduction step stabilizing the binding of the priming nucleotide was carried out after limited chain elongation. These results illustrate the conservative evolution of the active site of eukaryotic RNA polymerases.

; several of their structural genes have been cloned (Young and Davis, 1983;Allison et al., 1985;Riva et al., 1986) but little is known about the role of their multiple polypeptide components in RNA synthesis or about the functional homology of immunologically related polypeptides (Sentenac, 1985).
In the experiments described in this communication, RNA polymerase A, B, and C from yeast were first reacted with a nonradioactive derivative of ATP esterified at its terminal phosphate group with 4-formylphenol (Grachev et al., 1986b). The aldehyde group is expected to react reversibly (Venegas et al., 1973) with nucleophilic amino groups of the protein, and the Schiff base formed can be stabilized by reduction with sodium borohydride. Specific labeling occurs when active RNA polymerase molecules, containing in their active center the nucleotide derivative in the correct configuration, catalyze phosphodiester bond formation with a radioactive ribonucleoside triphosphate. The product of the enzymatic reaction was found attached exclusively to the second largest subunit of A, B, and C RNA polymerases. Fig. 1 shows the affinity labeling of yeast RNA polymerases A, B, and C and the identification of the labeled subunit by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In each case, a single band was labeled which corresponds to the second largest subunit BI50, A135, or C128,1 respectively ( Fig. 1,  lanes 1). Several controls demonstrated the selectivity of this affinity labeling. No discrete labeled band was detected when the ATP derivative was omitted or replaced by ATP or in the absence of template. Labeling of the BlsQ subunit was inhibited by a-amanitin (Fig. 2). The a-amanitin sensitivity of the labeling reaction was the same as that exhibited by yeast RNA polymerase B in a standard RNA synthesis reaction (i.e. 50 and 90% inhibition at 1 and 20 pg/ml of toxin, respectively). Labeling of the AI,, subunit of RNA polymerase A was strongly reduced by a-amanitin at high concentrations (0.4-2 mgjml) (Fig. 1,lane 8) in keeping with the weak sensitivity of enzyme A to this toxin (Huet et al., 1975). These observations clearly show that this affinity labeling is not due to a nonenzymatic radiolabeling of protein (Schmidt and Hanna, 1986).

RESULTS
The presence of 0.5 mM ATP during incubation inhibited the labeling of enzyme A but to a lesser extent that of BIso or Clz8 (Fig. 1, lanes 7). A 10-fold excess of ATP was required to diminish the labeling of enzyme B (Fig. 2 ) or enzyme C (not shown). When comparing the three enzymes we found that enzyme B reacted most efficiently with the 4-formylphenyl ester of ATP. However, the labeling of enzyme A was strongly increased and that of enzyme B decreased when using the 2formylphenyl ester of ATP (not shown). With all derivatives tested, the second largest subunit of the three enzymes was always the only labeled polypeptide. The radioactivity incorporated was resistant to treatments of the transcription complex by DNase I and RNase A but was sensitive to proteinase K. For characterization of the labeled RNA product, the radioactive subunit B,,, was eluted The polypeptide components of the RNA polymerases are designated by letter indicating the enzyme form from which it derives and a subscript corresponding to its molecular mass x lo+. , and C were prepared as described (Buhler et al., 1974;DezglCe et al., 1972;Huet et al., 1985). 4-Formylphenyl and the 2-formylphenylester of ATP were prepared as described (Grachev et al., 1986b(Grachev et al., , 1987. For purification of the product, the pH of the solution was adjusted to pH 7-8 with 1 M Tris base. After addition of 2.5 units of calf intestinal phosphatase (Boehringer Mannheim), the mixture was incubated for 90 min a t 37 "C to cleave unreacted ATP. The reaction product was purified by chromatography on a 2.5 X 0.5-cm DEAE cellulose column with triethylammoniumhydrogencarbonate, pH 8, and dried in uacuo. The RNA polymerases (1.5-2 pg) were incubated for 30 min a t 30 "C in 9 p1 of a mixture containing 0.5 mM of the ATP derivative, 50 mM Hepes'/ NaOH, pH 7.9, 1 mM dithiothreitol, 5 mM MgCl2, and 2.5 mM MnCI2 (enzyme A) or 1.5 mM MnC12 (enzyme C). For the B enzyme the buffer contained, in addition to the derivative, 10 mM Hepes/NaOH, pH 7.9,75 mM KCl, 1 mM dithiothreitol, and 2.5 mM MnCI2. Then 1 pl of 100 mM NaBH, was added and the incubation was continued for 30 min. The modified enzyme was labeled by initiating RNA synthesis with 0.1 p~ [a-"PIUTP (3000 Ci/ mmol) in the presence of DNA (1 pg) for 30 min. The template was denatured calf thymus DNA for RNA polymerases A and B or poly[d(A-T)] (Boehringer Mannheim) for enzyme C. Except when indicated the samples were treated for 20 min a t 30 ' C with RNase A (8 pg/ml) and DNase I (8 pg/ml) prior to electrophoresis on a sodium dodecyl sulfate-polyacrylamide slab gel. When indicated, proteinase K (Merck, Darmstadt) treatment was for 30 min a t 30 "C with 40 pg/ml of enzyme. Protein bands were silver-stained (lune A, B, and C ) and subjected to autoradiography using Kodak X-Omat S film and Kodak X-Omat C intensifying screen at -70 "C. Lane I , complete system; lune 2, without DNase and RNase treatment; lune 3, ATP derivative omitted; lune 4, ATP instead of ATP derivative; lune 5, DNA template omitted; lune 6, proteinase K treatment; lune 7, incubation with equimolar amounts of ATP and ATP derivative; lune 8, a-amanitin (2 mg/ml) during RNA synthesis. For RNA polymerase C samples the nuclease treatment was omitted, except in lune 2", treatment with DNase I, and lune 9, with RNase A. Lanes A, B, and C correspond to silver-stained RNA polymerases A, B, and C, respectively. Molecular mass ( X of subunits is indicated. from the gel (Grachev et al., 1986a) and degraded with proteinase K in 10 mM NH4HC03, pH 7.8, for 3 h at 37 "C. After inactivation of proteinase K by phenylmethylsulfonyl fluoride, the product was cleaved by acid pyrophosphatase from tobacco and phosphatase, similar to the procedure described by D' Allessio (1982). Upon chromatographic analysis (Mosig  et al., 1985), 44% of the total radioactivity migrated with the same rate as the trinucleotide ApApU in four different separating systems; 27% exhibited the mobility of authentic ApU. Its sequence was confirmed by specific enzymatic hydrolysis (Mosig et al., 1985). 9% migrated like free phosphate, and 'The abbreviation used is: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
20% of the radioactivity remained at the origin. The formation of the trinucleotide product indicates that yeast enzyme B is capable of catalyzing the formation of two phosphodiester bonds while having the priming nucleotide covalently attached to the active site.
The stable fixation of the radioactive product to RNA polymerase may also be carried out after the enzymatic reaction . Under these conditions, both large subunits became labeled upon incubating the ATP derivative with [cY-~'P]UTP and various combinations of nucleoside triphosphates (none, G T P or C T P + GTP, 1 p~ each) (Fig. 3). One migrated with the rate of BIs0 and one slightly above Bls as seen by superimposing the stained gel and the autoradiography. Both bands were sensitive to pro-

Labeling of RNA Polymerases from
Yeast 14379 a-Ama. pg/ml ATPmM Left (R), yeast RNA polymerase B (2.1 pg) was incubated for 15 min a t 30 'C with the ATP derivative, and then for 30 min in the presence of [a-:'*P]UTP, G T P (1 p~ each), and DNA template. Then the reduction with NaBH. was carried out as described in Fig. 1. Lane 1 tease and resistant to DNase (Fig. 3B, lanes 2 and 3). However, the radioactive band migrating slightly above BIR5 was partially degraded by RNase (Fig. 3B, lane 4). In this case larger RNA chains may be partially accessible to RNase attack. Analysis of the RNA chains linked to this subunit showed a polydisperse distribution of size in the range of 10-25 nucleotides. The binding of an RNA product larger than a trinucleotide probably explains the retarded migration of the largest subunit as recently observed by Bartholomew et al. (1986). When a similar experiment was carried out with enzyme A, one radioactive band migrated slightly above subunit Aln5 and another one above AIw (Fig. 3A). The labeling of B,, or AIw was a-amanitin-sensitive and was not observed when the ATP derivative, the template, or the reduction step were omitted or when the Schiff base was reduced prior to the addition of the nucleotides (data not shown). These results show that after start the nascent RNA chain is not attached to any of the smaller subunits but only to the two largest ones.

DISCUSSION
The first important conclusion which can be derived from the affinity labeling of the three forms of yeast RNA polymerase is that in each case the second largest subunit, i.e. B150, Alns, or Clns, interacts with the priming modified nucleoside triphosphate. Since the labeling procedure requires the catalytic activity of the enzymes, it achieves the unambiguous labeling of the active site. Here the definition of active site also includes a mobile part of the polypeptide backbone which can reach close enough to the amino acids involved in the catalytic process. The second conclusion is that the second largest subunit of enzymes A, B, and C are functionally homologous. They are also homologous in function to the subunit B140 from wheat germ (Grachev et al., 1986a) and to the @-subunit of E. coli RNA polymerase (Smirnov et al., 1981;Grachev et al., 1987). This conclusion is in agreement with previous immunological studies that revealed cross-reaction between yeast B150, A13s, and ClnR (Sentenac, 1985) and between yeast BIN and wheat germ B140 (Huet et d., 1982). The structural and functional homology of the second largest subunit of yeast RNA polymerases confirms the previous contention that the three nuclear RNA polymerases must be derived from a common ancestral enzyme after triplication of the genes for the large subunits (Sentenac and Hall, 1982).
Analysis of the oligoribonucleotides covalently attached to RNA polymerase B revealed the presence of the dinucleotide ApU and a large proportion of the trinucleotide ApApU. The elongation of the bound priming nucleotide by an adenine nucleotide in the absence of added ATP may be due to a trace impurity of ATP present in the preparation of y-substituted ribonucleoside triphosphate. Furthermore, it has been observed that the modified nucleoside triphosphates can serve weakly as substrates for the RNA polymerase from E. coli (Grachev et al., 1980) and for the yeast enzyme B in a poly[d(A-T)]-directed reaction (data not shown). The synthesis of the trinucleotide raises the question of the topology of the polymerase active site. If the attached dinucleotide remains locked in the active site the binding of the third nucleotide and the formation of a second phosphodiester bond implies the existence of a second, functionally equivalent active site (Panka and Dennis, 1985). Alternatively, the translocation of the active center could possibly occur if there is sufficient flexibility of the polypeptide backbone and/or of the spacer attaching the priming nucleotide.
In view of the synthesis of a sizeable proportion of ApU covalently bound to subunit Bls,, it is interesting to note that a-amanitin suppressed more than 90% this labeling. The same was found for the enzyme B from wheat germ (Grachev et al., 1986a). This indicates that the toxin inhibits the formation of the first phosphodiester bond and not only the translocation step as has been proposed for the enzyme from calf thymus (Vaisius and Wieland, 1982).
Since the largest subunit of eukaryotic RNA polymerase B has been associated with the sensitivity to a-amanitin (Greenleaf, 1983) as well as with the process of chain elongation (Coulter and Greenleaf, 1985;Ruet et al., 1980), it is conceivable that both large subunits contribute to the active site in a broad sense ( i e . including initiation and translocation). This notion is supported by our observation that after limited elongation the largest subunit also becomes labeled. The Grachev, M. A., Lukhtanov, E. A., and Mustaev, A. A. (198613)  Huet, J., Buhler, J.-M., Sentenac, A., and Fromageot, P. (1975) Proc.