346
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[36]
Blue staining profile of membrane proteins separated by sodium dodecyl sulfate gel electrophoresis. Additional problems include the screening of ultraviolet light by high nucleotide concentration or possible enzymic conversion of 8-N~cAMP to 8-N3-AMP with subsequent labeling of AMP binding sites. Each protein photolabeled with a reagent, such as with [3~P]8-N3-cAMP, must be checked with several experiments to ensure that it is indeed specific for both the natural compound and its biological mimic.
[35] 5 - F o r m y l - U T P for D N A - D e p e n d e n t R N A P o l y m e r a s e By Vic ARMSTRONG, HANS STERNBACH, a n d FRITZ ECKSTEIN In order to determine which subunits of DNA-dependent RNApolymerase contain the catalytic site, affinity labeling has been employed by various groups. 1-5 We have synthesized 5-formyluridine 5'-triphosphate (f@UTP) for this purpose. Such a derivative should be able to function as an affinity label by reaction with an amino group at the active
JO4..CHO
OH OH OH ~ H (1)
o
(]I) 1V. W. Armstrong, H. Sternbach, and F. Eckstein, Biochemistry 15, 2086 (1976). 2 A. M. Frischauf and K. H. scheit, Biochem. Biophys. Res. Commun. 53, 1227 (1973). 3j. Nixon, T. Spoor, J. Evans, and A. Kimball, Biochemistry 11, 4570 (1972). 417. Y.-H. Wu and C.-W. Wu, Biochemistry 13~ 2562 (1974). 5 p. Bull, J. Zaldivar, A. Venegas, J. Martial, and P. Valenzuela, Biochem. Biophys. Res. Commun. 64, 1152 (1975).
[36]
UTP FOR DNA-DEPENDENT RNA POLYMERASE
347
site of the enzyme to form a Schiff base. Subsequent reduction with sodium borohydride would then covalently link the triphosphate to the enzyme. However, during the synthesis of this triphosphate, a base-catalyzed anomerization6,7 occurred such that the final product was a mixture of the a-(l) and fl-(2) anomers. The anomers could be separated chromatographically with DEAE-Sephadex. The a-anomer has been used successfully to affinity-label RNA polymerase whereas the fl-anomer is a substrate for the enzyme. S y n t h e s i s of a- and ~ - f o s U T P
The required functionality is introduced into the 5 position of the uracil ring by hydroxymethylation of 2',3'-isopropylideneuridines followed by oxidation of the hydroxymethyl derivative with active Mn029 to yield 5-formyl-2',3'-isopropylideneuridine. The latter is converted into the mono- and triphosphate by conventional procedures. During the synthesis of the monophosphate by the method of Tener, 1° a base-catalyzed anomerization occurs so that a mixture of the a- and fl-anomers of 5-formyluridine monophosphate is produced. After conversion to the triphosphate, the two anomeric triphosphates may be separated. 5-Hydroxymethyl-2P,8'-O-isopropylideneuridine
8
2',3',-Isopropylideneuridine (24 g, 0.10 mole) is dissolved in a mixture of 200 ml of 0.5 N potassium hydroxide and 76 ml of (35%) formaldehyde. The solution is heated at 50 ° until reaction is complete (usually 4-6 hr). After allowing the solution to cool to room temperature, it is neutralized with Merck-I (H + form) ion-exchange resin. The resin is removed by filtration and washed well with methanol-water (1:1, v / v ) ; the filtrate and washing are combined and evaporated under reduced pressure. The resulting syrup is dissolved in chloroform, and the solution is applied to a silica-gel column (50 X 8 cm). The column is first washed with chloroform-methanol (19:1, v/v) until excess formaldehyde is eluted. It is then washed with chloroform-methanol (8:2, v/v), and the fractions 6V. W. Armstrong and F. Eckstein, Nucl. Acids Res. Suppl. 1, 97 (1975). V. W. Armstrong, J. K. Dattagupta, F. Eckstein, and W. Saenger, Nucl. Acids Res. 3, 1791 (1976). K. H. Scheit, Chem. Ber. 99, 3884 (1966). 9j. Attenburrow, A. F. B. Cameron, J. H. Chapman, R. M. Evans, t~. A. Hems, A. B. A. Jansen, and T. Walker, J. Chem. Soc. 1952, 1094 (1952). 10G. M. Tener, J. Am. Chem. Soc. 83, 159 (1961).
348
~.NZYMES, ANTIBODIES, AND OTHER PROTEINS
[35]
containing 5-hydroxymethyl-2",3"-isopropylideneuridine, as determined by thin-layer chromatography (TLC), are collected and evaporated under reduced pressure to yield 22.5 g of the product as a foam. Since this material is homogeneous by TLC and nuclear magnetic resonance (NMR), it may be used directly in the next step.
5-F orm yl-2',3'-O-isopropyl ideneuridine To 5-hydroxymethyl-2',3'-isopropylideneuridine (20 g, 64 mmoles) in dichloromethane (500 ml) is added active manganese dioxide 9 (100 g), and the suspension is stirred at room temperature for 48 hr. It is filtered over Celite with the aid of a suction pump and washed well with chloroform-methanol (1:1, v/v). The filtrate and washings are combined and evaporated under reduced pressure. The residue is dissolved in chloroform and applied to a silica gel colmnn (50 X 6 cm) which is eluted with 1 liter of chloroform, 1.5 liters of chloroform-methanol (98:2, v/v), and then with chloroform-methanol (96:4, v/v). Fractions of 200 ml are collected and are monitored by TLC. Those fractions containing pure 5-formyl2',3'-isopropylideneuridine are combined and evaporated to dryness under reduced pressure to yield 7.2 g of the desired compound as a white crystalline residue, m.p. 157°-159 °, hm m° 280 nm (13,600) and 235 nm (9000). This material is sufficiently pure for most synthetic purposes. It may be recrystallized from ethanol-water (95:5, v/v), m.p. 160°-161°.
Synthesis of the Mixture of ~- and fl-5-Formyluridine 5'-Monophosphates 5-Formyl-2',3'-isopropylideneuridine (2 mmoles) are treated with 4 mmoles of fl-cyanoethyl phosphate as described by Tener 1° but using 6 mmoles of triisopropylbenzenesulfonyl chloride instead of dicyclohexylcarbodiimide; this change reduces the reaction time to 4 hr. Hydrolysis of the cyanoethyl p~otecting group is carried out in 4 N N a O H - M e O H (1:1, v/v) for 30 min. Finally, the isopropylidene protecting group is cleaved with 50% aqueous acetic acid at 100 ° for 2 hr. The monophosphate is purified over a Dowe× 1 X 4 (100-200 mesh, C1-) column (40 X 4 cm), eluted with a linear gradient of 0.05 M LiCI-0.01 M HCI and 0.15 M LiC1-0.01 M HC1 (2 liters of each). The product elutes between 0.07 and 0.09 M LiC1 and is detectable by the ratio of its UV absorption at 280 nm to that at 260 nm (approximately 2.00). The fractions containing 5-formyIuridine 5'-monophosphate are pooled and neutralized with 1 M LiOH. They are concentrated under reduced pressure to approximately 20 ml, and the solution is divided between two 50-ml centrifuge tubes. Saturated BaC12 solution, 5 drops, is added to each tube
[36]
UTP FOR DN&-DEPENDENT RNA POLYMERASE
349
followed by 40 ml of ethanol. After eentrifugation, the supernatant liquid is decanted and the residue is washed with 30 ml of 70% aqueous ethanol. The barium salt of 5-formyluridine 5'-monophosphate is converted into its Na* salt by stirring with Merek-I (Na + form) ion-exchange resin. After filtering the resin and washing with water, the filtrate and washings are evaporated under reduced pressure to yield 10,500 A~_sounits of 5-formyluridine 5'-monophosphate. The product gives a single spot on electrophoresis at pH 7.5 with a mobility of 12.8 cm (UMP, 13.2 cm). A single spot (Rs = 0.21) is obtained after paper chromatography in ethanol/1 M ammonium acetate (7:3, v/v).
Synthesis of a- and fl-foSUTP Of the anomeric mixture of fo'~UMP, 10,500 A..~,, units are converted to their triphosphates by a standard procedure2 ~ The crude reaction product is charged onto a column of DEAE-Sephadex and eluted with a linear gradient of 2.5 liters each of 0.05 M and 0.45 M triethylammonium bicarbonate. The a-fosUTP (856 A~so units) elutes between 0.39 and 0.42 M buffer and the fl-anomer (1190 A~o units) between 0.35 and 0.38 M buffer. These structures have been assigned after comparison of their CD and N M R spectra to spectra of the corresponding nucleosides. For enzymic studies, these compounds may be purified further by passage over Dowex ion-exchange resin as described for [,/-~P]-a-fo~UTP below.
Synthesis of [7-~2P]a-fosUTP This derivative is prepared essentially according to the method of Glynn and Chappell. 1~ a-fosUTP, 150 A2so units, is incubated in a final volume of 1.2 ml containing 0.2 ml 1 M Tris-chloride at pH 8.0, 12 ~1 of 1 M MgCl~, 0.2 ml of 0.1 M NaOH, 3 ~1 of 0.1 M Na2HPO~, 20 ~1 of 3-phosphoglycerate (cyclohexylammonium salt, 20 mg/ml), 20 f~l of glyceraldehyde-3-phosphate dehydrogenase (10 mg/ml), 12 ~1 of phosphoglycerate kinase (10 mg/ml), and 0.1 ml of ~2Pi (10 mCi/ml, carrier free). After 16 hr at room temperature, the mixture is diluted with 1 ml of water and chromatograhed over a Dowex 1 X 4 (200-400 mesh, C1-) ion-exchange column (10 X 0.08 cm) with a linear gradient of 0.01 M HC1-0.05 M LiC1 and 0.01 M HC1-0.45 M LiC1 (180 ml of each). The fractions containing a-fo.~UTP are collected and neutralized with 1 M LiOH. After concentration under reduced pressure to approximately 5 ml, the solution is transferred to a centrifuge tube. Saturated BaCI=, solution, " A. M. Michelson, Biochim. Biophys. Acta 91, 1 (1964). ~ I. M. Glynn and J. B. Chappell, Biochem. J. 90, 147 (1974).
350
ENZrMES, ANTIBODIES, AND OTHER PROTEINS
[36]
5 drops, is added followed by 30 ml of EtOH to precipitate the barium salt of the triphosphate. After centrifugation, the supernatant liquid is decanted and the residue is washed twice with 2 ml of water by centrifugation. Finally, the triphosphate is converted to its Na ÷ salt with Mcrck-I (Na ÷ form) ion-exchange resin, and, after evaporation under reduced pressure, the a-fosUTP is dissolved in 1 ml of H20 and stored as a frozen solution at --20 °. Yield = 92 A28o units. 280/260 = 1.98. Specific activity = 2.34 X 108 cpm/mole.
Enzyme Purification and Assay Escherichia coli RNA-polymerase holo- and core enzymes is were 95% pure by sodium dodecyl sulfate (SDS) gel electrophoresis. Enzyme activity was measured as the amount of [14CLAMP or [I~C]UMP incorporated into acid-insoluble material 14 after a 10-min incubation at 37 °. The assay mixture contained in 0.1 ml 40 mM Tris-chloride at pH 8.0, 8 mM MgCl2, 5 mM dithioerythritol, 0.2 A~6o unit of poly[d(AT)], 0.05 M KCI, 1 mM ATP, and 1 mM [14C]UTP. For kinetic studies, a fixed concentration of ATP (0.4 mM) was used and the concentration of [I~C]UTP was varied. Enzyme activity was measured as the amount of [1~C] UTP incorporated into acid-insoluble material after 5 min. Inhibition o] RNA Polymerase by a-]oSUTP a-fosUTP is a noncompetitive inhibitor of the poly [d (AT) ] directed synthesis by RNA polymerase with a Kl -- 0.54 mM. The fl-anomer, on the other hand, proved to be a substrate for the enzyme with a K m = 0.12 mM [Kin (UTP) -- 0.05 mM]. The inhibition of RNA polymerase by a a-fo"UTP is dependent upon the a-fo~UTP concentration, and for maximum inhibition at any particular concentration a short preincubation of enzyme and a-foSUTP is required (Fig. 1). It is important that the a-fosUTP concentration in the assay solution is the same as that in the preincubation solution, since on dilution of the a-fosUTP the enzyme regains activity with time (Fig. 1) indicating that the inhibition is reversible.
~odium Borohydride-Induced Irreversible Inhibition Upon reduction of a preincubated enzyme/a-fosUTP solution with an equal volume of 0.1 M sodium borohydride solution, the enzyme was irreversibly inhibited. The extent of the inhibition was dependent upon is R. R. Burgess, J. Biol. Chem. 244, 6168 (1969). ~ F. J. Bollum, Proc. Nucl. Acid Res. 1, 296 (1966).
[36]
UTP
FOR
1°°t
X
DNA-DEPENDENT
X..
X
.,
O~ ~ #s i 'A
RNA X
351
POLYMERASE )4,
X
-0"
¢
~,6C
/
/.0 LU
20
o
Ib
2b
2%
Preincubation time (min)
Fie. 1. The inhibition of P~NA polymerase by a-foSUTP. The enzyme was preincubated at 37° in a reaction mixture containing 40 mM Tris-chloride, pit 8.0, 8 mM MgCh, 0.05 M KCI and (a) tt,O ( X ~ X ) , (b) 0.23 mM a-fo~UTP (A. A), and (c) 0.46 mM a-fo~UTP ( O ~ O ) After various times, 10-/~l aliquots were removed and assayed. In a further experiment (d) ((D---C)) 10-#1 aliquots were removed from (c) after 5-min preincubation and diluted with a solution (70 /~l) containing 57.1 mM Tris-chloride, pH 8.0, llA mM MgCL, 7.1 mM DTE, 0.071 M KC1, 1 ram ATP, and 1 mM [I*C]UTP. The incubation was continued at 37°, and after various times the enzyme was assayed by the addition of a solution (20 #1) containing 0.2 A~eounit of poly[d(AT)]. the a-fosUTP concentration, reaching 80% inhibition at 2 m M a-fosUTP. T o test the specificity of this inhibition, the nucleoside a-fosU was also used. The inhibition in this case was about half that produced by the triphosphate, reaching only 42% at 2 m M a-fo~U. The B-analogs were also tested and found to be inferior to a-fosUTP. At 2 raM, fl-fosUTP produced 35% and fl-fosU 24% inhibition after NaBH4 reduction. Further evidence for the specificity of the binding of a-fosUTP was provided by protection experiments with ATP, U T P , and GTP. Addition of these triphosphates to the preincubation mixture of a-fosUTP and R N A polymerase reduced the extent of the inhibition.
Stoichiometry of Binding o/a-]osUTP and Location o] Its Binding Site(s) The amount of [~_s2p]_a_fosUTP, prepared by a previously published procedure/~ covalently linked to the enzyme after sodium borohydride
352
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[35]
reduction was determined by retention of the protein on nitrocellulose membrane filters. The amount of label retained on the filters increased with the time of the preincubation of enzyme and [),-s2P]a-fosUTP. However, during the same period no significant increase in the inhibition af the enzyme occurred. After a 20-sec preincubation the stoichiometry of binding was 1.1:1 ([~,-32P]a-fosUTP bound:inactivated enzyme) and after 10 rain it had risen to 2.42:1. The location of the label after a 20-see preincubation was determined by separating the enzyme into its different subunits by electrophoresis in 6 M urea on cellulose acetate plates. 1' Most of the label was found to run with the fl subunit. This subunit has been shown to be the site of rifampicin action,~6, ~7 and other affinity labeling studies 2-4 have implicated this subunit as containing at least part of the catalyic center of RNA polymerase. Comments
The data presented here were obtained with holoenzyme. Core enzyme gave similar results indicating that a factor has no influence on the binding of a-fosUTP. Poly[d (AT)] also had no effect. It had originally been our intention to use fl-fosUTP as an affinity label for RNA polymerase. However, this triphosphate was a substrate for the enzyme and only produced weak inhibition on reduction of an enzyme-fl-fosUTP mixture. On the other hand, a-fo5UTP proved to be a potent inhibitor of the enzyme, and we therefore concentrated our effort on this analog. Although this no longer has the usual fl configuration of those triphosphates that are substrates for RNA-polymerase, Rhodes and Chamberlin is have shown that the elongation site in the ternary complex has a general affinity for the triphosphate moiety. The inhibition of RNA polymerase by the binding of a-fosUTP to the fl subunit has been shown to satisfy several criteria for affinity labeling: (1) it forms a noncovalent complex prior to covalent attachment; (2) the inhibition and covalent attachment of a-fo~UTP can be suppressed by the presence of nucleoside triphosphates; (3) the triphosphate a-fosUTP is a more potent inhibitor than the nucleoside a-fosU; and (4) the a-fosUTP stoichiometrically labels RNA polymerase (after a 20-sec preincubation). D. Rabussay and W. Zillig, FEBS Left. 5, 104 (1969). ~6W. Zillig, K. Zechel, D. Rabussay, M. Schachner, V. Sethi, P. Palm, A. Heil, and W. Seifert, Cold Spring Harbor Symp. Quart. Biol. 35, 47 (1970). 17W. Stender, A. A. Stiitz, and K. H. Scheit, Eur. J. Biochem. 56, 129 (1975). ~8G. Rhodes and M. J. Charaberlin, ]. Biol. Chem. 249, 6675 (1974).
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[36]
Blue staining profile of membrane proteins separated by sodium dodecyl sulfate gel electrophoresis. Additional problems include the screening of ultraviolet light by high nucleotide concentration or possible enzymic conversion of 8-N~cAMP to 8-N3-AMP with subsequent labeling of AMP binding sites. Each protein photolabeled with a reagent, such as with [3~P]8-N3-cAMP, must be checked with several experiments to ensure that it is indeed specific for both the natural compound and its biological mimic.
[35] 5 - F o r m y l - U T P for D N A - D e p e n d e n t R N A P o l y m e r a s e By Vic ARMSTRONG, HANS STERNBACH, a n d FRITZ ECKSTEIN In order to determine which subunits of DNA-dependent RNApolymerase contain the catalytic site, affinity labeling has been employed by various groups. 1-5 We have synthesized 5-formyluridine 5'-triphosphate (f@UTP) for this purpose. Such a derivative should be able to function as an affinity label by reaction with an amino group at the active
JO4..CHO
OH OH OH ~ H (1)
o
(]I) 1V. W. Armstrong, H. Sternbach, and F. Eckstein, Biochemistry 15, 2086 (1976). 2 A. M. Frischauf and K. H. scheit, Biochem. Biophys. Res. Commun. 53, 1227 (1973). 3j. Nixon, T. Spoor, J. Evans, and A. Kimball, Biochemistry 11, 4570 (1972). 417. Y.-H. Wu and C.-W. Wu, Biochemistry 13~ 2562 (1974). 5 p. Bull, J. Zaldivar, A. Venegas, J. Martial, and P. Valenzuela, Biochem. Biophys. Res. Commun. 64, 1152 (1975).
[36]
UTP FOR DNA-DEPENDENT RNA POLYMERASE
347
site of the enzyme to form a Schiff base. Subsequent reduction with sodium borohydride would then covalently link the triphosphate to the enzyme. However, during the synthesis of this triphosphate, a base-catalyzed anomerization6,7 occurred such that the final product was a mixture of the a-(l) and fl-(2) anomers. The anomers could be separated chromatographically with DEAE-Sephadex. The a-anomer has been used successfully to affinity-label RNA polymerase whereas the fl-anomer is a substrate for the enzyme. S y n t h e s i s of a- and ~ - f o s U T P
The required functionality is introduced into the 5 position of the uracil ring by hydroxymethylation of 2',3'-isopropylideneuridines followed by oxidation of the hydroxymethyl derivative with active Mn029 to yield 5-formyl-2',3'-isopropylideneuridine. The latter is converted into the mono- and triphosphate by conventional procedures. During the synthesis of the monophosphate by the method of Tener, 1° a base-catalyzed anomerization occurs so that a mixture of the a- and fl-anomers of 5-formyluridine monophosphate is produced. After conversion to the triphosphate, the two anomeric triphosphates may be separated. 5-Hydroxymethyl-2P,8'-O-isopropylideneuridine
8
2',3',-Isopropylideneuridine (24 g, 0.10 mole) is dissolved in a mixture of 200 ml of 0.5 N potassium hydroxide and 76 ml of (35%) formaldehyde. The solution is heated at 50 ° until reaction is complete (usually 4-6 hr). After allowing the solution to cool to room temperature, it is neutralized with Merck-I (H + form) ion-exchange resin. The resin is removed by filtration and washed well with methanol-water (1:1, v / v ) ; the filtrate and washing are combined and evaporated under reduced pressure. The resulting syrup is dissolved in chloroform, and the solution is applied to a silica-gel column (50 X 8 cm). The column is first washed with chloroform-methanol (19:1, v/v) until excess formaldehyde is eluted. It is then washed with chloroform-methanol (8:2, v/v), and the fractions 6V. W. Armstrong and F. Eckstein, Nucl. Acids Res. Suppl. 1, 97 (1975). V. W. Armstrong, J. K. Dattagupta, F. Eckstein, and W. Saenger, Nucl. Acids Res. 3, 1791 (1976). K. H. Scheit, Chem. Ber. 99, 3884 (1966). 9j. Attenburrow, A. F. B. Cameron, J. H. Chapman, R. M. Evans, t~. A. Hems, A. B. A. Jansen, and T. Walker, J. Chem. Soc. 1952, 1094 (1952). 10G. M. Tener, J. Am. Chem. Soc. 83, 159 (1961).
348
~.NZYMES, ANTIBODIES, AND OTHER PROTEINS
[35]
containing 5-hydroxymethyl-2",3"-isopropylideneuridine, as determined by thin-layer chromatography (TLC), are collected and evaporated under reduced pressure to yield 22.5 g of the product as a foam. Since this material is homogeneous by TLC and nuclear magnetic resonance (NMR), it may be used directly in the next step.
5-F orm yl-2',3'-O-isopropyl ideneuridine To 5-hydroxymethyl-2',3'-isopropylideneuridine (20 g, 64 mmoles) in dichloromethane (500 ml) is added active manganese dioxide 9 (100 g), and the suspension is stirred at room temperature for 48 hr. It is filtered over Celite with the aid of a suction pump and washed well with chloroform-methanol (1:1, v/v). The filtrate and washings are combined and evaporated under reduced pressure. The residue is dissolved in chloroform and applied to a silica gel colmnn (50 X 6 cm) which is eluted with 1 liter of chloroform, 1.5 liters of chloroform-methanol (98:2, v/v), and then with chloroform-methanol (96:4, v/v). Fractions of 200 ml are collected and are monitored by TLC. Those fractions containing pure 5-formyl2',3'-isopropylideneuridine are combined and evaporated to dryness under reduced pressure to yield 7.2 g of the desired compound as a white crystalline residue, m.p. 157°-159 °, hm m° 280 nm (13,600) and 235 nm (9000). This material is sufficiently pure for most synthetic purposes. It may be recrystallized from ethanol-water (95:5, v/v), m.p. 160°-161°.
Synthesis of the Mixture of ~- and fl-5-Formyluridine 5'-Monophosphates 5-Formyl-2',3'-isopropylideneuridine (2 mmoles) are treated with 4 mmoles of fl-cyanoethyl phosphate as described by Tener 1° but using 6 mmoles of triisopropylbenzenesulfonyl chloride instead of dicyclohexylcarbodiimide; this change reduces the reaction time to 4 hr. Hydrolysis of the cyanoethyl p~otecting group is carried out in 4 N N a O H - M e O H (1:1, v/v) for 30 min. Finally, the isopropylidene protecting group is cleaved with 50% aqueous acetic acid at 100 ° for 2 hr. The monophosphate is purified over a Dowe× 1 X 4 (100-200 mesh, C1-) column (40 X 4 cm), eluted with a linear gradient of 0.05 M LiCI-0.01 M HCI and 0.15 M LiC1-0.01 M HC1 (2 liters of each). The product elutes between 0.07 and 0.09 M LiC1 and is detectable by the ratio of its UV absorption at 280 nm to that at 260 nm (approximately 2.00). The fractions containing 5-formyIuridine 5'-monophosphate are pooled and neutralized with 1 M LiOH. They are concentrated under reduced pressure to approximately 20 ml, and the solution is divided between two 50-ml centrifuge tubes. Saturated BaC12 solution, 5 drops, is added to each tube
[36]
UTP FOR DN&-DEPENDENT RNA POLYMERASE
349
followed by 40 ml of ethanol. After eentrifugation, the supernatant liquid is decanted and the residue is washed with 30 ml of 70% aqueous ethanol. The barium salt of 5-formyluridine 5'-monophosphate is converted into its Na* salt by stirring with Merek-I (Na + form) ion-exchange resin. After filtering the resin and washing with water, the filtrate and washings are evaporated under reduced pressure to yield 10,500 A~_sounits of 5-formyluridine 5'-monophosphate. The product gives a single spot on electrophoresis at pH 7.5 with a mobility of 12.8 cm (UMP, 13.2 cm). A single spot (Rs = 0.21) is obtained after paper chromatography in ethanol/1 M ammonium acetate (7:3, v/v).
Synthesis of a- and fl-foSUTP Of the anomeric mixture of fo'~UMP, 10,500 A..~,, units are converted to their triphosphates by a standard procedure2 ~ The crude reaction product is charged onto a column of DEAE-Sephadex and eluted with a linear gradient of 2.5 liters each of 0.05 M and 0.45 M triethylammonium bicarbonate. The a-fosUTP (856 A~so units) elutes between 0.39 and 0.42 M buffer and the fl-anomer (1190 A~o units) between 0.35 and 0.38 M buffer. These structures have been assigned after comparison of their CD and N M R spectra to spectra of the corresponding nucleosides. For enzymic studies, these compounds may be purified further by passage over Dowex ion-exchange resin as described for [,/-~P]-a-fo~UTP below.
Synthesis of [7-~2P]a-fosUTP This derivative is prepared essentially according to the method of Glynn and Chappell. 1~ a-fosUTP, 150 A2so units, is incubated in a final volume of 1.2 ml containing 0.2 ml 1 M Tris-chloride at pH 8.0, 12 ~1 of 1 M MgCl~, 0.2 ml of 0.1 M NaOH, 3 ~1 of 0.1 M Na2HPO~, 20 ~1 of 3-phosphoglycerate (cyclohexylammonium salt, 20 mg/ml), 20 f~l of glyceraldehyde-3-phosphate dehydrogenase (10 mg/ml), 12 ~1 of phosphoglycerate kinase (10 mg/ml), and 0.1 ml of ~2Pi (10 mCi/ml, carrier free). After 16 hr at room temperature, the mixture is diluted with 1 ml of water and chromatograhed over a Dowex 1 X 4 (200-400 mesh, C1-) ion-exchange column (10 X 0.08 cm) with a linear gradient of 0.01 M HC1-0.05 M LiC1 and 0.01 M HC1-0.45 M LiC1 (180 ml of each). The fractions containing a-fo.~UTP are collected and neutralized with 1 M LiOH. After concentration under reduced pressure to approximately 5 ml, the solution is transferred to a centrifuge tube. Saturated BaCI=, solution, " A. M. Michelson, Biochim. Biophys. Acta 91, 1 (1964). ~ I. M. Glynn and J. B. Chappell, Biochem. J. 90, 147 (1974).
350
ENZrMES, ANTIBODIES, AND OTHER PROTEINS
[36]
5 drops, is added followed by 30 ml of EtOH to precipitate the barium salt of the triphosphate. After centrifugation, the supernatant liquid is decanted and the residue is washed twice with 2 ml of water by centrifugation. Finally, the triphosphate is converted to its Na ÷ salt with Mcrck-I (Na ÷ form) ion-exchange resin, and, after evaporation under reduced pressure, the a-fosUTP is dissolved in 1 ml of H20 and stored as a frozen solution at --20 °. Yield = 92 A28o units. 280/260 = 1.98. Specific activity = 2.34 X 108 cpm/mole.
Enzyme Purification and Assay Escherichia coli RNA-polymerase holo- and core enzymes is were 95% pure by sodium dodecyl sulfate (SDS) gel electrophoresis. Enzyme activity was measured as the amount of [14CLAMP or [I~C]UMP incorporated into acid-insoluble material 14 after a 10-min incubation at 37 °. The assay mixture contained in 0.1 ml 40 mM Tris-chloride at pH 8.0, 8 mM MgCl2, 5 mM dithioerythritol, 0.2 A~6o unit of poly[d(AT)], 0.05 M KCI, 1 mM ATP, and 1 mM [14C]UTP. For kinetic studies, a fixed concentration of ATP (0.4 mM) was used and the concentration of [I~C]UTP was varied. Enzyme activity was measured as the amount of [1~C] UTP incorporated into acid-insoluble material after 5 min. Inhibition o] RNA Polymerase by a-]oSUTP a-fosUTP is a noncompetitive inhibitor of the poly [d (AT) ] directed synthesis by RNA polymerase with a Kl -- 0.54 mM. The fl-anomer, on the other hand, proved to be a substrate for the enzyme with a K m = 0.12 mM [Kin (UTP) -- 0.05 mM]. The inhibition of RNA polymerase by a a-fo"UTP is dependent upon the a-fo~UTP concentration, and for maximum inhibition at any particular concentration a short preincubation of enzyme and a-foSUTP is required (Fig. 1). It is important that the a-fosUTP concentration in the assay solution is the same as that in the preincubation solution, since on dilution of the a-fosUTP the enzyme regains activity with time (Fig. 1) indicating that the inhibition is reversible.
~odium Borohydride-Induced Irreversible Inhibition Upon reduction of a preincubated enzyme/a-fosUTP solution with an equal volume of 0.1 M sodium borohydride solution, the enzyme was irreversibly inhibited. The extent of the inhibition was dependent upon is R. R. Burgess, J. Biol. Chem. 244, 6168 (1969). ~ F. J. Bollum, Proc. Nucl. Acid Res. 1, 296 (1966).
[36]
UTP
FOR
1°°t
X
DNA-DEPENDENT
X..
X
.,
O~ ~ #s i 'A
RNA X
351
POLYMERASE )4,
X
-0"
¢
~,6C
/
/.0 LU
20
o
Ib
2b
2%
Preincubation time (min)
Fie. 1. The inhibition of P~NA polymerase by a-foSUTP. The enzyme was preincubated at 37° in a reaction mixture containing 40 mM Tris-chloride, pit 8.0, 8 mM MgCh, 0.05 M KCI and (a) tt,O ( X ~ X ) , (b) 0.23 mM a-fo~UTP (A. A), and (c) 0.46 mM a-fo~UTP ( O ~ O ) After various times, 10-/~l aliquots were removed and assayed. In a further experiment (d) ((D---C)) 10-#1 aliquots were removed from (c) after 5-min preincubation and diluted with a solution (70 /~l) containing 57.1 mM Tris-chloride, pH 8.0, llA mM MgCL, 7.1 mM DTE, 0.071 M KC1, 1 ram ATP, and 1 mM [I*C]UTP. The incubation was continued at 37°, and after various times the enzyme was assayed by the addition of a solution (20 #1) containing 0.2 A~eounit of poly[d(AT)]. the a-fosUTP concentration, reaching 80% inhibition at 2 m M a-fosUTP. T o test the specificity of this inhibition, the nucleoside a-fosU was also used. The inhibition in this case was about half that produced by the triphosphate, reaching only 42% at 2 m M a-fo~U. The B-analogs were also tested and found to be inferior to a-fosUTP. At 2 raM, fl-fosUTP produced 35% and fl-fosU 24% inhibition after NaBH4 reduction. Further evidence for the specificity of the binding of a-fosUTP was provided by protection experiments with ATP, U T P , and GTP. Addition of these triphosphates to the preincubation mixture of a-fosUTP and R N A polymerase reduced the extent of the inhibition.
Stoichiometry of Binding o/a-]osUTP and Location o] Its Binding Site(s) The amount of [~_s2p]_a_fosUTP, prepared by a previously published procedure/~ covalently linked to the enzyme after sodium borohydride
352
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[35]
reduction was determined by retention of the protein on nitrocellulose membrane filters. The amount of label retained on the filters increased with the time of the preincubation of enzyme and [),-s2P]a-fosUTP. However, during the same period no significant increase in the inhibition af the enzyme occurred. After a 20-sec preincubation the stoichiometry of binding was 1.1:1 ([~,-32P]a-fosUTP bound:inactivated enzyme) and after 10 rain it had risen to 2.42:1. The location of the label after a 20-see preincubation was determined by separating the enzyme into its different subunits by electrophoresis in 6 M urea on cellulose acetate plates. 1' Most of the label was found to run with the fl subunit. This subunit has been shown to be the site of rifampicin action,~6, ~7 and other affinity labeling studies 2-4 have implicated this subunit as containing at least part of the catalyic center of RNA polymerase. Comments
The data presented here were obtained with holoenzyme. Core enzyme gave similar results indicating that a factor has no influence on the binding of a-fosUTP. Poly[d (AT)] also had no effect. It had originally been our intention to use fl-fosUTP as an affinity label for RNA polymerase. However, this triphosphate was a substrate for the enzyme and only produced weak inhibition on reduction of an enzyme-fl-fosUTP mixture. On the other hand, a-fo5UTP proved to be a potent inhibitor of the enzyme, and we therefore concentrated our effort on this analog. Although this no longer has the usual fl configuration of those triphosphates that are substrates for RNA-polymerase, Rhodes and Chamberlin is have shown that the elongation site in the ternary complex has a general affinity for the triphosphate moiety. The inhibition of RNA polymerase by the binding of a-fosUTP to the fl subunit has been shown to satisfy several criteria for affinity labeling: (1) it forms a noncovalent complex prior to covalent attachment; (2) the inhibition and covalent attachment of a-fo~UTP can be suppressed by the presence of nucleoside triphosphates; (3) the triphosphate a-fosUTP is a more potent inhibitor than the nucleoside a-fosU; and (4) the a-fosUTP stoichiometrically labels RNA polymerase (after a 20-sec preincubation). D. Rabussay and W. Zillig, FEBS Left. 5, 104 (1969). ~6W. Zillig, K. Zechel, D. Rabussay, M. Schachner, V. Sethi, P. Palm, A. Heil, and W. Seifert, Cold Spring Harbor Symp. Quart. Biol. 35, 47 (1970). 17W. Stender, A. A. Stiitz, and K. H. Scheit, Eur. J. Biochem. 56, 129 (1975). ~8G. Rhodes and M. J. Charaberlin, ]. Biol. Chem. 249, 6675 (1974).
