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The effects of several divalent cations on the activation or inhibition of RNA polymerases II
ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 203, No. 2, September, pp. 553-564, 1980
The Effects of Several Divalent Cations on the Activation Inhibition of RNA Polymerases II1 ALAIN Department
C. VAISIUF
AND
qf Botany, University Mississauga,
Ontario
or
PAUL A. HORGEN
of Toronto, Erindale LSL lC6, Canada
Campus,
Received October 18. 1979 Eukaryotic RNA polymerases II or B have been generally shown to be more active with MnZ+rather than Mg*+ as the required divalent metal ion. However, very little was known about the activation or inhibition of RNA polymerases by metal ions other than Mg2+and MnZ+. In this study the effects of several divalent cations were observed on in vitro transcription by highly purified RNA polymerases II from the mushroom Agaricus bisporus and calfthymus. Cobalt (II), Fez+, Mg2+, or Mn*+ may function as the divalent metal activator for RNA synthesis catalyzed by RNA polymerase II from calfthymus with the maximal activity observed using Fe’+ as the activating metal ion. Of the 13divalent cations used in this study, only Co2+ or Mn*+ can serve as the required divalent metal for RNA polymerase II from A. bisporus. Barium (II) had an apparent synergistic effect on the activation of RNA polymerase II from A. bisporus and calfthymus, and SF+ exhibited a strong positive synergism on the activity ofthe mushroom enzyme. RNA polymerases II from both the mushroom and calf thymus were significantly inhibited by Be’+, CdY+,Hg’+, Ni2+, and Zn’+. A. bisporus RNA polymerase II was strongly inhibited by Cu2+and Fez+. The particularly potent metal inhibitor, Zn*+, was shown to manifest competitive inhibition for both enzymes with respect to the substrates as determined with Mn-uridine triphosphate. Zinc (II) was also shown to manifest mixed inhibition with respect to the DNA template cofactor for both the calf thymus and mushroom RNA polymerase II.
DNA-dependent RNA polymerases (ribonucleosidetriphosphate; RNA-nucleotidyltransferase, E.C. 2.7.7.6) require an added divalent cation for activation. It has almost become axiomatic to state that eukaryotic RNA polymerases II are significantly more activated by Mn’+ rather than Mg2+,whereas RNA polymerases I and III are almost equally stimulated by Mn2+ or Mg*+ (l-3). However, very little is known about the activation or inhibition of RNA polymerases by metal ions other than Mg*+ and Mn2+ despite the fact that the involvement of various metal ions in the transcriptional process can occur through an interaction with the substrates, the template cofactor, the RNA
polymerase molecule, as well as with the binary and ternary complexes (4-12). Considering the possible complexity of interactions, the effects of various metal ions on RNA synthesis catalyzed by RNA polymerase might also be expected to be quite varied. Although it had been presumed that divalent metal ions were required in transcription as chelate complexes with the nucleoside triphosphates which then became the true substrates for RNA synthesis, there was little evidence suggesting the specific function of the metal ion activator with RNA polymerase. Recently, Mildvan and his coworkers used Mn’+ as a paramagnetic probe to demonstrate that E. coli RNA polymerase ’ This work was supported in part by National Rehas a single tight binding site for the metal search Council of Canada Grant A6938. * To whom correspondence should be sent at the ion cofactor at the catalytic elongation site (10-12). Also, it was determined that there present address:Max-Planck-Institut fur medizinische Forschung, Abteilung Naturstoff Chemie, D-6900 were six weaker binding sites of Mn*+ on Heidelberg, West Germany. the E. coli RNA polymerase molecule but 553
0003-9861/80/100553-12$02.00/O Copyright 0 1980by AcademicPress, Inc. All rights of reproductionin any form reserved.
554
VAISIUS AND HORGEN
the role of these weaker binding sites remains unknown (10). The fact that Mn2+ is not bound at the initiation site (10, 12) does not preclude the possibility of the activating metal ion functioning in the initiation reaction. The requirement for a metal activator at initiation has been clearly demonstrated (13, 14) using an abortive initiation reaction (15) to determine the parameters for the formation of the first phosphodiester bond. As yet, a similar tight binding site for the metal ion activator has not been demonstrated for any eukaryotic RNA polymerase. Perhaps significantly with regard to the complexity which might be expected with eukaryotic RNA polymerases, it was shown that Mn2+ and Mg2+ had distinctly differing effects on the initiation reaction as well as on the elongation reaction of RNA synthesis catalyzed by RNA polymerase I (16). Metal ions are likely to participate in transcription through an interaction with nucleic acids, and it is likely that these interactions will confer variable degrees of specificity on transcription depending on the template and the metal ion. For example, it was reported that RNA polymerase II from calf thymus would form a stable complex with superhelical SV40 DNA form I in the presence of Mn2+ but would not form a similar stable complex in the presence of Mg2+ (1’7). RNA polymerase I from yeast preferentially transcribes ribosomal cistrons with Mg2+ as a cofactor, whereas the same enzyme will randomly transcribe the template with MnZ+ as a cofactor (18). Again, it should be emphasized that little is known regarding the specific effects of metal ions other than Mg2+ or Mn2+ on the association of RNA polymerase with the template despite the fact that many divalent metal ions are known to interact quite differently with a number of reaction sites on nucleic acids (4-8). Since there is little doubt that metal ions play a vital role in the biological process of transcription, it becomes relevant to consider the possible effects of various metal ions on in vitro transcription. There is ample evidence suggesting that many physiologically important metal ions as well as those exhibiting mutagenic effects will accumulate in the cell nucleus (20-25). Because there
was a dearth of information regarding the effects of metal ions other than Mg2+or Mn2+ on in vitro transcription, we have examined the effects of several divalent cations on the activation and inhibition of highly purified RNA polymerases II from the mushroom. A. bisporus and from calf thymus. We expect this information to be useful in future studies of the transcriptional process. MATERIALS
AND METHODS
Chemicals. Calf thymus DNA (Type 1) and nucleoside triphosphates were purchased from Sigma. Tritiumlabeled UTP (37.5 Wmmol) and [3H]CTP (27.8 Ci/ mmol) were purchased from New England Nuclear. Spectroscopically pure manganese chloride and magnesium chloride were purchased from Johnson Matthey Chemicals. All other metals were the highest purity chloride or sulfate salts obtainable from Baker Chemical Company or from Fisher Scientific. Protein detwmin4hms. Protein concentrations were estimated using the Bradford method with prepared reagents from Bio-Rad Laboratories (26). Purijication of RNA polymerase II. All buffers used throughout these experiments were prepared with degassed, deionized water double distilled in glass. RNA polymerase II resistant to a-amanitin from the mushroom A. bisporus was prepared as previously described (27). The purification procedures for RNA polymerase II from calf thymus were similarly modified from Jendrisak and Burgess (28). The purification procedures involes precipitation with polyethylenimine, selective elution of RNA polymerase II from the polyethylenimine precipitate, ammonium sulfate fractionation, batch adsorption and elution with DEAE-cellulose, DEAESephadex chromatography, P-cellulose chromatography, and exclusion chromatography on Bio-Gel agarose, 1.5 M. As shown in Fig. 1, RNA polymerases II from calf thymus and from A. bisporus were purified to near homogeneity as evidenced by polyacrylamide gel electrophoresis under nondenaturing conditions. RNA polymerase assay. The standard assay solution was 75 ~1 having 7% (v/v) glycerol; 75 mM (NH&SO,; 10 mM Tris-HCl (pH 7.9); 0.4 mM each of ATP, CTP, and GTP; 1 &i of [SH]UTP (1 &i/3 nmol); and 5 pg of denatured calf thymus DNA. The concentration of the divalent cation and 2-mercaptoethanol are indicated in the appropriate table or figure legend. In those control assays measuring dependency upon UTP, the labeled substrate was [3H]CTP (1 &i/3 nmol). The concentration and purity of the substrates and template were determined spectroscopically. In all assays without 2mercaptoethanol, the reactions were prepared and carried out under nitrogen. Spectroscopicdeterminations verified that Fe*+ was oxidized in the presence of 2mercaptoethanol, whereas it was not if the reaction so-
EFFECTS
OF DIVALENT
CATIONS
l&ions were incubated under nitrogen in the absence of Z-mercaptoethanol. The enzyme and the template were allowed to preincubate with the metal ions prior to the addition of the substrates. The assay solutions were then incubated for 15 min at 30°C. The reaction was terminated with the addition of 25 ~1 of a solution 16% (w/v) sodium pyrophosphate at 3O”C, and a predetermined aliquot was spotted on a DE-81 (Whatman) disk. The disks were pooled and washed five times for 10 min each time in 0.25 M Na,HP04 (20 ml/disk). The disks were then twice washed in 95% ethanol, and allowed to dry. Radioactivity was counted in a 2:l mixture of PCS (Amersham/ Searle)-xylene scintillation fluid. Corrections were made for the relative counting efficiencies of [“H]UTP and (:‘H]UMP incorporated into RNA as previously described (29). One unit of RNA polymerase activity is defined as the incorporation of 1 nmol of labeled nucleotide into RNA per 15 min at 30°C. Kinetic experiments for Zn 2+inhibition. The effects of Zn2+ on the kinetics of RNA synthesis were determined with a varying substrate concentration. The standard assay conditions were modified as follows: the concentration of ATP, CTP, and GTP were fixed at 0.4 mM with [:jH]CTP (1 pCi/lO nmol) as the labeled nucleotide; the concentration of UTP was varied (0.025, 0.05, 0.075, and 0.10 mM) for each inhibitor concentration and the reactions were incubated for 7.5 min at 25°C. Under these assay conditions, incorporation of the labeled nucleotide always remained less than 2% of the UTP concentration. The effects of Zn*+ on the kinetics of RNA synthesis were determined for varying concentrations of the necessary template cofactor. In these experiments, the standard assay conditions were followed with the exceptions that the DNA concentration was varied ( 1, 2.5, 7.5, 15, and 25 pg/ml) for each inhibitor concentration and that the reactions were incubated for 7.5 min at 25°C. In separate experiments, it was determined that the rate of RNA synthesis was linear for each concentration of DNA used and that the highest concentration used was not saturating. All data were the result of two separate experiments using duplicate assays in each. The best fit lines were determined by use of preprogrammed linear regression analysis.
ON RNA POLYMERASES
555
II
CALFTHYMUS
0
2
6
6
4
Distance ( cm )
A. BISPORUS
P
0.2
I 0.1
2
LAO
2
k
6
6
’
Distance ( cm ) FIG. 1. Polyacrylamide gel electrophoresis under nondenaturing conditions of purified RNA polymerases II from calf thymus and A. bisporus. Electrophoresis was carried out as described by Davis (48). The gels shown are 5% acrylamide and were stained using the method of Blakesley and Boezi (49).
factors in transcription. Reagent grade Mg2+ will serve as a poor activator of RNA polymerase II from A. bisporus whereas ultrapure Mg2+ will ellicit only negligible activity. Using ultrapure Mg2+ with calf thymus RNA polymerase II will cause a decrease in activity but some activity was always measurable under our standard assay conditions. As shown in Table I, all metals tested were done so in the presence and absence of the RESULTS reducing agent 2-mercaptoethanol. This was Several divalent cations were tested as necessary since 2-mercaptoethanol readily activators of transcription catalyzed by RNA complexes with many heavy metals to form polymerase II from calf thymus and A. bis- mercaptides, which could have the apparent porus. As shown in Table I, calf thymus effect of removing metal ions from solution, RNA polymerase II can use Co*+, Fez+, or could facilitate the interaction of free nucleotides with nucleic acids (30). It should Mg2+, and Mn2+as cofactors in transcription. Likewise, RNA polymerase II from A. bis- be noted that highly variable results were porus can use Co2+ as well as Mn2+ as co- obtained with either Cu2+ or Fe2+ as a
556
VAISIUS AND HORGEN TABLE I
THEEFFECTSOFDIVALENTCATIONSASACTIVATORS OFTRANSCRIPTIONBY RNA POLYMERASESIIFROM Agaricus bisporus AND CALF THYMUS~ Specific activity (unitsimg protein) Metal salt (2 mM)
2-Mercaptoethanol (1 mM)
A. bisporus
Calf thymus
BaCl,
+
7.3 0.4
2.0 0
BeSO
+ -
0 0
2.0 0
CdCl,
+ -
0 0
0 0
CaCl,
+ -
0 0
0 0
CoCl,
+ -
49.1 28.6
77.4 74.0
cuso*
+ -
? 0
? 0
FeSO,
+
0 0
? 168.7
MgCl,
+ -
12.5 5.5
23.7 11.3
MnCl, (ultrapure)
+ -
86.2 44.8
125.8 72.9
HgCl,
+ -
NiCl,
+ -
SrCl,
+ -
ZnSO,
+ -
U In those assays without 2-mercaptoethanol, the reactions were carried out under nitrogen.
potential activator if the enzyme was not assayed under nitrogen and in the absence of 2-mercaptoethanol. In Table I, the results obtained for CL?+ or Fez+ in the presence of 2-mercaptoethanol were listed as questionable because the percentage standard deviation for six assays with each metal ion exceeded 10% (normally only values of 5%
or less were tolerated) and control assays for Cu2+ and Fez+ incubated without enzyme were as much as 20 times higher than control assays for the other metals (normal control values were 50-150 cpm). As shown in Fig. 2, titrations of RNA polymerase II from either A. bisporus or calf thymus with Mn2+ exhibit similar activation profiles. There is a slight difference in the concentration of Mn2+ which yields optimal activity with a standardized assay, as the optimal concentration of Mn2+ is 0.8 mM for calf thymus and 1.75 mM for A. bisporus. Quite similar activation profiles are obtained for both enzymes with Co2+as the divalent cation. When calf thymus RNA polymerase II is titrated with Fe2+, a broad activation profile is observed with optimal activity near 4.5 111M.A. bisporus is not even minimally activated by Fe2+ over concentrations ranging from 0.2 to 20 11IM. Tables II and III include several controls which were seen to further substantiate the activation of RNA polymerases II by the metals listed and to exclude the possibility of aberrant catalytic activity with these metals. Assays were run in the absence of a template and in the absence of each of the nucleotides to determine whether or not the metals might catalyze the random polymerization of polyribonucleotides through terminal addition, or catalyze the unprimed synthesis of complementary polyribonucleotides (31, 32). With regard to Fe2+, it was thought that Fe2+ might serve as a reducing agent which very efficiently facilitates the activity of RNA polymerase and the actual activator metal ion might be Mn’+ or another metal ion which remained tightly bound to the enzyme through the isolation procedures. Although this factor cannot be completely dismissed, it seems quite unlikely since the enzyme is not even minimally activated in the absence of an added metal ion over a time course up to 2.5 h. The a-amanitin control further substantiates that the incorporation is catalyzed by RNA polymerases II, and that other metal ion activators do not alter the inhibition by a-amanitin. Because multiple forms of RNA polymerase have yet to be purified from the mushroom and thus their inhibition patterns are unknown, the results of the a-amanitin control are less certain for
EFFECTS OF DIVALENT
CATIONS ON RNA POLYMERASES
557
II
50
25
0 1.0
2.0
3.0
4.0
5.0
6.0
7.0
6.0
9.0
* 0
METAL ION (mM) FIG. 2. (A) The activation of calf thymus RNA polymerase II by divalent metal ions Mn*+ (O), Co2+ (O), M@+(D), and Fe*+ (Cl). (B) The activation of RNA polymerase II from A. bisporus by divalent metal ions Mn2+(O), and Co2+(0). In those assays with Co2+and Fe *+, 2-mercaptoethanol was omitted from the reaction solution and the reactions were carried out under nitrogen.
RNA polymerase II from A. bisporus, which RNA polymerase II. Calf thymus RNA polyis half-maximally inhibited by cw-amanitinat merase II was strongly inhibited by Cu2+ a concentration of 6.5 pg/ml (27), but con- but only at concentrations approaching those tamination with another form of RNA poly- of the metal activator. merase or some other nucleotidyl transferase The inhibition by Mg2+ and the slight inseems unlikely considering the purity of the hibition evidenced with Co2+may not accuenzyme preparations. rately be inhibition but rather a case of As shown in Tables IV and V, several di- these metals functioning as activators but valent cations were shown to be inhibitors with the reaction proceeding much more of RNA polymerase II from the mushroom slowly even in the presence of Mn2+. Also, or calf thymus. Both enzymes were signifi- it can be seen in Tables IV and V that Ba2+ cantly inhibited by Be2+, Cd2+, Hg2+, Ni2+, had an apparent synergistic effect on the and Zn2+ at a concentration lo-fold less than activation of RNA polymerase II from A. bithe optimal concentration of Mn2+which was sporus and calf thymus, and SP exhibited used as the metal activator. Copper (II) and a strong positive synergism on the activity Fe2+were also strong inhibitors ofA. bispom of the mushroom enzyme. As previously
558
VAISIUS TABLE
AND HORGEN
II
REQUISITEASSAYCOMPONENTSFORTHEINCORPORATION OF LABELED NUCLEOTIDE INTO RNA UNDER STANDARDASSAYCONDITIONSBYRNAPOLYMERASE II FROMAgaricus bisporus USING THE OPTIMAL CONCENTRATIONOFEITHERCO~+ORM~~+AS AN ACTIVATOR Specific activity (unitsimg protein) CoCl,
MnCl,
Reaction conditions
(1.5 mM)
(1.75 mM)
Complete standard assay solution - Enzyme -Metal activator -DNA -ATP -CTP -GTP -UTP +a-Amanitin (100 pg/ml)
46.0 0 0 0.07 0.21 0.53 0.49 0.18 6.4
102.5 0 0 0.02 0.26 0.27 0.19 0.09 1.6
inhibition by Zn*+ of RNA polymerases II from both. A. bisporus and calf thymus. Using the above data, the apparent Ki for the inhibition of MnUTP incorporation by Zn*+ (ZnNTP) was calculated to be 0.0027 lllM for RNA polymerase II from A. bisporus and 0.0320 mM for RNA polymerase II from calf thymus. Replots of the data by the Eadie-Hofstee method (35) did not detect any deviation from linearity, thus confirming the observation that inhibition by Zn*+ was apparently competitive. Because the above plots might not clearly differentiate partial competition, Dixon (36) has suggested a method of analysis which would discern partially competitive inhibition. If the reciprocal of the fractional inhibition is plotted against the reciprocal of the inhibitor concentration, the intercept will be unity for pure competitive inhibition whereas the intercept will be greater than unity for partially competitive inhibition (36). Using this method of analysis, the intercepts for curves with each substrate concentration were very near to unity for the experiments using either the mushroom (& = 1.86 + 15%) or the calf thymus (c? = 0.36 ? 21%) enzyme.
stated in a multicomponent reaction such as the RNA polymerase assay, the free metal ion will have a number of potential binding sites, e.g., template, nascent RNA nucleotides, and SO:*. Thus, the apparent synerTABLE III gistic effects of Ba*+ and Sr*+ could result from the displacement of Mn+* from such REQUISITEASSAYCOMPONENTSFORTHEINCORPORAsites where it does not have any direct effect TION OF LABELED NUCLEOTIDE INTO RNA UNDER STANDARDASSAYCONDITIONSBYRNAPOLYMERASE on RNA polymerase. The nature of Zn*+ inhibition on the re- IIFROMCALFTHYMUSUSINGTHEOPTIMALCONCENTRATION OF EITHER Co2+, Fez+, OR MnL+ AS action kinetics of RNA polymerases II were AN ACTIVATOR determined with a varying concentration of the substrate UTP. From calculations using Specific activity the reported stability constants of Mn*+ with (units/mg protein) nucleoside triphosphates (33, 34), it can be reasonably assumed that the true substrates CoCl, MnCI, Reaction FeS04 (4.5 mM) (0.8 mM) (1 mM) are MnNTP3 complexes under the assay conditions conditions employed in this study. It is also quite likely that the true inhibitor is a ZnNTP Complete standard 140.4 68.7 171.6 assay solution complex. Because Mn*+ will bind SOY?with a 0 0 0 -Enzyme KD of 5.0 mM, Zn*+ inhibition might be ex0 0 0 -Metal pected to be dependent upon the SO:’ con- -DNA 0.04 0.05 0.04 centration. As shown in Table VI, Zn*+ -ATP 0.16 0.25 0.09 inhibition increases with an increasing con- -CTP 0.35 0.33 0.29 centration of (NH,),SO,. Graphical analysis -GTP 0.68 0.07 0 of the data by Lineweaver-Burk plots as -UTP 0.12 0.15 0 +cu-Amanitin shown in Fig. 3 demonstrates competitive (0.5 @g/ml) B Abbreviation
used: NTP, nucleoside triphosphate.
2.01
0.51
0.50
EFFECTS OF DIVALENT
CATIONS ON RNA POLYMERASES
Because an inhibitor may also effect the binding or functioning of a cofactor, the effects of Zn2+ inhibition were observed with varying concentrations of the template cofactor. As shown in Fig. 4, Lineweaver-Burk plots indicate an apparent mixed inhibition
THE EFFECTS OF DIVALENT CATIONS AS INHIBITORS BY RNA POLYMERASE OF IN VITRO TRANSCRIPTION II FROM Agaricus bisporus”
559
TABLE V THE EFFECTS
OF DIVALENT
CATIONS
OF IN VITRO TRANSCRIPTION
BY
II FROM CALF
RNA
AS INHIBITORS POLYMERASE
THYMUF
Concentration (mM)
Specific activity (unitslmg protein)
Fractional inhibition
BaCl,
0.1 1.0
132.1 144.5
None None
BeSO*
0.1 1.0
88.4 6.3
0.329 0.952
Metal salt
TABLE IV
II
Concentration (mM)
Specific activity (unitsimg protein)
Fractional inhibition
CdCl,
0.1 1.0
84.2 3.2
0.361 0.976
BaCI,
0.2 2.0
101.6 143.7
None None
CaCl,
0.1 1.0
137.6 116.8
None 0.114
BeSO,
0.2 2.0
32.7 3.4
0.639 0.962
CoClB
0.1 1.0
87.6 78.4
None 0.090
CdCl,
0.2 2.0
11.7 1.4
0.871 0.985
cuso,*
0.1 1.0
81.8 2.9
0.051 0.966
CaCl,
0.2 2.0
90.0 73.4
0.007 0.190
FeSOt
0.1 1.0
104.7 177.9
None None
coc1:
0.2 2.0
42.7 30.5
0.197 0.427
WZ
0.1 1.0
114.7 83.2
0.130 0.369
cuso*4
0.2 2.0
1.2 0.1
0.977 0.999
HgCl;
0.1 1.0
60.5 1.6
0.298 0.981
FeSO<
0.2 2.0
26.3 2.1
0.505 0.960
NiCl,
0.1 1.0
67.4 5.8
0.489 0.956
MgCl,
0.2 2.0
88.8 57.8
0.020 0.362
SrCI,
0.1 1.0
104.7 76.3
0.206 0.421
HgCl$
0.2 2.0
1.3 0
0.976 1.00
ZnSO:
0.1 1.0
5.8 0.7
0.933 0.992
NiCl,
0.2 2.0
60.3 4.8
0.334 0.947
SrCl,
0.2 2.0
100.2 140.2
None None
ZnSO*,
0.2 2.0
1.7 0.1
0.968 0.998
a Under standard assay conditions with 1 mM Mn2+ as the metal activator and 1 mM 2-mercaptoethanol as the reducing agent, the specific activity was 131.8 unitsimg protein. If 2-mercaptoethanol was omitted, the specific activity was 86.2 units/mg protein. An asterisk (*I indicates assays without 2-mercaptoethanol. In those assays without 2-mercaptoethanol, the reactions were carried out under nitrogen.
Metal salt
” Under standard assay conditions with 2 mM Mn2+ as the metal activator and 1 mM 2-mercaptoethanol as the reducing agent, the specific activity was 90.6 units/mg protein. If 2-mercaptoethanol was omitted, the specific activity was 53.2 unitsimg protein. An asterisk (*) indicates the assays without 2-mercaptoethanol. In those assays without 2-mercaptoethanol, the reactions were carried out under nitrogen.
by Zn2+ with respect to the template cofactor for reactions catalyzed by RNA polymerase II from eitherd. bisporus or calfthymus. Zinc as well as other metal ions have been shown to be able to cause the depolymerization of ribonucleic acids (37-39). Although
560
VAISIUS AND HORGEN
merases II. Although the enzymes were isolated from widely divergent species, the THE EFFECTOF(NH,),SO,CONCENTRATION ONTHE RNA polymerases II from the mushroom and INHIBITION WITH 2.5 x lo-” M Zn2+ OF IN VITRO from calf thymus have a similar subunit comTRANSCRIPTION BY RNAPOLYMERASEII position (27, 40), i.e., insofar as can be disFROMCALFTHYMUS" cerned by SDS-polyacrylamide gel electroTABLE VI
WH,),SO, concentration (mM)
Fractional inhibition
0 25 50 75 100 125
0.147 0.158 0.164 0.175 0.227 0.228
(1The fractional inhibition was determined under standard assay conditions with 1 InM Mn*+ as the metal activator.
the reaction conditions are quite different for the depolymerization of RNA by Zn*+ than those used in our assays for RNA polymerase reactions, a control experiment was done to test whether or not the apparent inhibition could be related to depolymerization of the RNA product by Zn2+. The control experiment was as follows: triplicate samples of calf thymus RNA polymerase were assayed under the standard conditions previously described, a second series of samples were assayed as described except that after the alloted incubation period the reactions were terminated with cr-amanitin (10 pg/ml) and then further incubated at the identical time and temperature of the standard reaction, a third series of samples were assayed and terminated by a-amanitin with the simultaneous addition of 0.05 mM Zn*+, and the sampleswere further incubated under standard conditions. There was very good agreement in measured activity for all samples, thus indicating that zinc does not measurably degrade the RNA product under the assay conditions employed in this study. DISCUSSION
Because a comparative study had not previously been done for the divalent cation requirement of eukaryotic RNA polymerases, we have observed the effects of several divalent metal ions on two distinct RNA poly-
FIG. 3. The effect of Zn*+ on the kinetics of RNA synthesis as catalyzed by RNA polymerases II: Lineweaver-Burk plot with varying concentrations of UTP. (A) Lineweaver-Burk plot of the activity of RNA polymerase II from A. bisporus as determined with several concentrations of Zn2+: (W) no Zr?+, (0) 2.5 x lo-'M Zn2+, (0) 2.5 x 10-OM Zn*+, (0) 5.0 x lo-" M Zn'+. The assay conditions are described within the text with the concentration of MnCl, at 1.75 mM, and with the enzyme concentration at either 0.9 or 1.1 pg per reaction. (B) Lineweaver-Burk plot of the activity of RNA polymerase II from calf thymus as determined with several concentrations of Zn*? (a) no ZnZ+, (0)2.5 x 1O-5M Znzf, (0) 5.0 x 10m5 M Zn2+. The assay conditions are described within the text with the concentration of MnCl, at 0.8 mM,and with the enzyme concentration at 0.6 kg per reaction.
EFFECTS
OF DIVALENT
CATIONS
ON RNA POLYMERASES
II
561
[DNA~ pg/mL
1 [OnA]
FIG. 4. The effect of Zn’+ on the kinetics of RNA synthesis as catalyzed by RNA polymerases II: Lineweaver-Burk plot with varying concentrations of the DNA cofactor. (A) Lineweaver-Burk plot of the activity of RNA polymerase II from A. bisporus as determined with several concentrations of Zn2+: (m) no Zn”+, (0) 2.5 x 10-j M Zn2+, (0) 5.0 x 10e5 M Zn2+. The assay conditions are described within the text concentration at either 0.9 or 1.1 pg per reaction. (B) Lineweaver-Burk plot of the activity of RNA polymerase II from calf thymus as determined with several concentrations of Zn2+: (W) no Zn”‘, (0) 5.0 x IO-” M Zn’+, (0) 2.5 x 1O-5 M Zn2+, (0) 5.0 x lo-” M Zn’+. The assay conditions are described within the text with the concentration of MnCl, at 0.8 mM, and with the enzyme concentration at 0.6 pg per reaction.
phoresis. The enzymes share a similar chromatographic behavior on several different ion-exchange resins (l-3, 27, and our unpublished results) and are similar in the general assay conditions yielding optimal activity in vitro (l-3,27). The similarity in catalytic properties has allowed us to use a quite uniform assay for RNA polymerase activity throughout this study. A prominent difference between the two enzymes is the fact that RNA polymerase II from A. bisporus is 3250-fold more resistant to a-amanitin than is calf thymus RNA polymerase II as
determined in our laboratory using identical procedures for each enzyme preparation. A. bisporus was considered an interesting source of RNA polymerase II for these experiments for the additional reason that mushrooms are known to accumulate significant quantities of trace metals (41, 42). Calf thymus RNA polymerase II had the obvious advantage of being one of the best characterized eukaryotic RNA polymerases (l-3). Our results indicate that effects of several divalent cations are generally similar on RNA polymerases II from A. bisporus or
562
VAISIUS
AND
calf thymus. This is particularly evident with Be2+, Cd2+, Hg2+, and Znz+ all of which are strong inhibitors of RNA polymerase II from either calf thymus or A. bisporus. Zinc (II), which is a particularly potent inhibitor of both RNA polymerases, is recognized as an essential growth factor for all organisms (43), and is known to accumulate in the cell nucleus of calf thymus as well as other organisms (21,25). Additionally, nucleotidyl transferases including RNA polymerases have been recognized as zinc metalloenzymes (19,43). Thus, it seems particularly interesting that Zn2+ was shown to be a competitive inhibitor with respect to the substrate (MnUTP). In these experiments both the labeled nucleotide CTP and the nucleotide of varied concentration UTP have not been recognized as initiating nucleotides (44). Although the detailed mechanisms of catalysis are not known for the eukaryotic RNA polymerases, it is generally accepted that there is but a single elongation site and quite possibly a single site for the metal activator at the elongation site. It is very likely that the actual site of Zn2+ competition is the site of the metal activator. Of course, under the technological limitations of our experimental design it is not possible to determine the true free metal concentration or to distinguish between competition with the metal activator site and the nucleotide site if the metal ion is required for the binding of the substrate. Because Zn2+ is a competitive inhibitor with regard to the binding of the substrate, it does not preclude inhibition with respect to the DNA cofactor since the binding of Zn2+ may also alter binding within the ternary complex. The pattern of mixed inhibition, i.e., a competitive as well as a noncompetitive aspect to the inhibition by Zn2+, might be expected considering the complexity of interactions necessary for catalysis. For example, a speculative explanation for mixed inhibition might be that the binding of Zn2+causes a decreased affinity within the ternary complex such that a conformational change is prevented which might be necessary for the proper base pairing between the incoming nucleotide and the template. With such an explanation, competitive inhibition would still be observed with respect to the substrate because increasing the MnNTP con-
HORGEN
centration would decrease the availability of RNA polymerases to the Zn2+ (ZnNTP) inhibitor. Mixed inhibition could also result from the fact that a ZnNTP complex not only competes with substrate binding but also prevents the dissociation of the ternary complex. It should be emphasized that other methods of analyses will be necessary to elucidate the precise nature of the interactions involved in Zn2+ inhibition and to this end it is interesting that the nature of Zn2+ inhibition was similar for the RNA polymerases II from both A. bispoms and calf thymus. The fact that Co2+ and Mn2+ as well as Mg2+ for calf thymus can serve as activating divalent cations might be expected considering that these metals can also activate prokaryotic RNA polymerases as well as other nucleotidyl transferases (19, 45). A striking difference in the RNA polymerases II from calf thymus and A. bisporus is their response to Fe2+. RNA polymerase II from A. bispoms is strongly inhibited by Fe2+, while Fe2+ is the metal ion activator yielding optimal activity for calf thymus RNA polymerase II. These results are interesting in light of the fact that Fe2+/Fe3+is a significant metal ion within the cell nucleus of calf thymus, and in particular with the deoxyribonucleoproteins (20,25,46). The oxidation and reduction of Fe2+/Fe3+ has even been suggested as a possible regulatory mechanism in gene control (7,47). It must be emphasized that free Fe2+/Fe3+ is unlikely to be found in the cell nucleus, and especially unlikely in the concentrations required in this study. Because our experimental design has centered around the use of a quite typical in vitro assay of RNA polymerase, we do not wish to engage in extended speculation on our part attempting to attribute a physiological role for various metal ions with respect to RNA polymerase vis-a-vis the DNA template. However, since there is ample evidence suggesting that many metal ions will accumulate in the cell nucleus (20-25,46), we do think it prudent to consider in future studies the possible role of various metal ions in all aspects of the transcriptional process in eukaryotes. In fact, our preliminary results from a study in progress indicate that the divalent cation activator may influence the
EFFECTS
OF DIVALENT
CATIONS
fidelity of transcription by calf thymus RNA polymerase II on defined templates. In addition, kinetic analysis of transcription with various activating or inhibiting metal ions may prove important in elucidating the chemical properties of the metal ion necessary for RNA synthesis catalyzed by RNA polymerase. ACKNOWLEDGMENTS The authors are particularly thankful for the incisive and helpful criticism of the reviewers. We wish to express our gratitude to Heather MacLeod for her expert technical assistance. We wish to thank Drs. Loeb and Mildvan for sending up preprints of works in press. We are grateful to Drs. Ng and Cummins for helpful discussions during the course of this work. REFERENCES 1. CHAMBON, P. (1974) in The Enzymes (Boyer, P. D., ed.), Vol. 10, pp. 261-331, Academic Press, New York. 2. CHAMBON, P. (1975) An?lu. Rev. Biochem. 44, 613-638. 3. ROEDER, R. (1976) in RNA Polymerase (Losick, R., and Chamberlin, M., eds.), pp. 285-329, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 4. IZATT, R. M., CHRISTENSEN, J. J., AND RYTTING, J. H. (1971) Cheln. Rev. 71, 439-481. WESER, U. (1968) S’truc. Bonding 5, 41-67. EICHHORN, G. L., AND SHIN, Y. A. (1968) J. Amer. Chem. Sot. 90, 7323-7328. SISSOGFF, I., GRISVARD, J., AND GUILL~, E. (1976) Progr. Biophys. Mol. Biol. 31, 165-199. MILDVAN, A. S. (1970) in The Enzymes (Boyer, P. D., ed.), Vol. 2, pp. 445-536. Academic Press, New York. 9. Tu, A. T., AND HELLER, M. J. (1974) in Metal Ions in Biological Systems (Sigel, H., ed.), Vol. 1, pp. l-49, Dekker, New York. 10. KOREN, R., AND MILDVAN, A. S. (1977) Biochemistry 16, 241-249. 11. BEAN, B. L., KOREN, R., AND MILDVAN, A. S. (1977) Biochemistry 16, 3322-3333. 12. STEIN, P. J., AND MILDVAN, A. S. (1978) Biochemistry 17, 2675-2684. 13. SHEMYAKIN, M. F., MALYGIN, A. G., AND PATRUSHEV, L. I. (1978) FEBS Lett. 91, 253-256. 14. MCCLURE, W. R., CECH, C. L., AND JOHNSTON, D. E. (1978) J. Biol. Chem. 253, 8941-8948. 15. JOHNSTON, D. E., AND MCCLURE, W. R. (1976) in RNA Polymerase (Losick, R., and Chamberlin, M., eds.), pp. 413-428, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
ON RNA POLYMERASES
II
563
16. NAGAMINE, Y., MIZUNO, D., AND NATORI, S. (1978) Biochim. Biophys. Acta 519, 440-446. 17. HOSSENLOPP, P., OUDET, P., AND CHAMBON, P. (1974) Eur. J. Biochem. 41, 397-411. 18. HAGER, G., HOLLAND, M., VALENZUELA, P., WEINBERG, F., AND RUTTER, W. J. (1976) in RNA Polymerase (Losick, R., and Chamberlin, M., eds.), pp. 745-762, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 19. MILDVAN, A. S., AND LOEB, L. A. (1979) Crit. Rev. Biochenz. 6, 219-244. 20. WACKER, W., AND VALLEE, B. L. (1959) J. Biol. Chern. 234, 3257-3262. 21. WESER, U., AND BISCHOFF, E. (1970) Eur. J. Biochem. 12, 571-575. 22. WESER, U., AND BRAUER, H. (1970) Biochim. Biophys. Acta 204, 542-549. 23. VALLEE, B. L., AND ULMER, D. (1972)Annu. Rev. Biochem. 41, 91-128. 24. WEBB, M., HEATH, J. C., AND HOPKINS, T. (1972) Brit. J. Cancer 26, 274-278. 25. SKRINSKA, V. A., MESSINEO, L., TOWNS, R. L., AND PEARSON, K. H. (1978) Ezpetientia 34, 15-16. 26. BRADFORD, M. (1976)AnaZ. Biochem. 72,248-254. 27. VAISIUS, A. C., AND HORGEN, P. A. (1979) Biochemistq 18, 795-803. 28. JENDRISAK, J. J., AND BURGESS, R. R. (1975) Biochemistry 14, 4639-4645. 29. ROEDER, R. (1974) J. Biol. Chem. 249, 241-248. 30. FRIEDMAN, M. (1973) The Chemistry and Biochemistry of the Sullhydryl Group in Amino Acids, Peptides, and Proteins, Pergamon Braunschweig. 31. STECK, T. L., CAICUTS, M. J., AND WILSON, R. G. (1968) J. Biol. Chem. 243, 2769-2778. 32. SMITH, D. A., RATCLIFF, R. L., WILLIAMS, D. L., AND MARTINEZ, A. M. (1967) J. Biol. Chem. 242, 590-595. 33. WALAAS, E. (1958) Acta. Chemica. &and. 12, 528-536. 34. BOCK, R. M. (1960) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds.), Vol. 2, pp. 3-38, Academic Press, New York. 35. DOWD, J. E., AND RIGGS, D. S. (1965) J. Biol. Chem. 240, 863-869. 36. DIXON, J. L. (1963) Enzymes and Metabolic Inhibitors, Vol. 1, Academic Press, New York. 37. BUTZOW, J. J., AND EICHHORN, G. L. (1965) Biopolymers 3, 95-107. 38. EICHHORN, G. L., TARIEN, E., AND BUTZOW, J. J. (1971) Biochemistry 10, 2014-2019. 39. BUTZOW, J. J., AND EICHHORN, G. L. (1971) Biochemistry 10, 2019-2027. 40. KEDINGER, C., GISSINGER, F., AND CHAMBON, P. (1974) Eur. J. Biochem. 44, 421-436. 41. DOMSCH, K. H., GRABBE, K., AND FLECKEN-
564
42. 43. 44. 45.
VAISIUS
AND
STEIN, J. (1976) 2. PJEanzenernaehr. Bodenk. 4,487-501. BYRNE, A. R., RAVNIK, V., AND KOSTA, L. (1976) Ski. Total Environ. 6, 65-78. VALLEE, B. L. (1976) Trends Biochem. Sci. 1, 88-91. MEILHAC, M., AND CHAMBON, P. (1973) Eur. J. Biochem. 35, 454-463. Fox, C. F., AND WEISS, S. B. (1964) J. Biol. Chem. 239, 175-185.
HORGEN 46. SKRINSKA, V. A., MESSINEO, L., TOWNS, R., AND PEARSON, K. (1978)Znt. J. B&hem. 9,637-638. 47. MINCHENKOVA, L. E., AND IVANOV, V. I. (1967) Biopolymere 5, 615-625. 48. DAVIS, B. J. (1964) Ann N. Y. Acad. Sci. 121, 404-427. 49. BLAKESLEY, R., AND BOEZI, J. (1977) Anal. &em. 82, 580-582.
Bio-
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 203, No. 2, September, pp. 553-564, 1980
The Effects of Several Divalent Cations on the Activation Inhibition of RNA Polymerases II1 ALAIN Department
C. VAISIUF
AND
qf Botany, University Mississauga,
Ontario
or
PAUL A. HORGEN
of Toronto, Erindale LSL lC6, Canada
Campus,
Received October 18. 1979 Eukaryotic RNA polymerases II or B have been generally shown to be more active with MnZ+rather than Mg*+ as the required divalent metal ion. However, very little was known about the activation or inhibition of RNA polymerases by metal ions other than Mg2+and MnZ+. In this study the effects of several divalent cations were observed on in vitro transcription by highly purified RNA polymerases II from the mushroom Agaricus bisporus and calfthymus. Cobalt (II), Fez+, Mg2+, or Mn*+ may function as the divalent metal activator for RNA synthesis catalyzed by RNA polymerase II from calfthymus with the maximal activity observed using Fe’+ as the activating metal ion. Of the 13divalent cations used in this study, only Co2+ or Mn*+ can serve as the required divalent metal for RNA polymerase II from A. bisporus. Barium (II) had an apparent synergistic effect on the activation of RNA polymerase II from A. bisporus and calfthymus, and SF+ exhibited a strong positive synergism on the activity ofthe mushroom enzyme. RNA polymerases II from both the mushroom and calf thymus were significantly inhibited by Be’+, CdY+,Hg’+, Ni2+, and Zn’+. A. bisporus RNA polymerase II was strongly inhibited by Cu2+and Fez+. The particularly potent metal inhibitor, Zn*+, was shown to manifest competitive inhibition for both enzymes with respect to the substrates as determined with Mn-uridine triphosphate. Zinc (II) was also shown to manifest mixed inhibition with respect to the DNA template cofactor for both the calf thymus and mushroom RNA polymerase II.
DNA-dependent RNA polymerases (ribonucleosidetriphosphate; RNA-nucleotidyltransferase, E.C. 2.7.7.6) require an added divalent cation for activation. It has almost become axiomatic to state that eukaryotic RNA polymerases II are significantly more activated by Mn’+ rather than Mg2+,whereas RNA polymerases I and III are almost equally stimulated by Mn2+ or Mg*+ (l-3). However, very little is known about the activation or inhibition of RNA polymerases by metal ions other than Mg*+ and Mn2+ despite the fact that the involvement of various metal ions in the transcriptional process can occur through an interaction with the substrates, the template cofactor, the RNA
polymerase molecule, as well as with the binary and ternary complexes (4-12). Considering the possible complexity of interactions, the effects of various metal ions on RNA synthesis catalyzed by RNA polymerase might also be expected to be quite varied. Although it had been presumed that divalent metal ions were required in transcription as chelate complexes with the nucleoside triphosphates which then became the true substrates for RNA synthesis, there was little evidence suggesting the specific function of the metal ion activator with RNA polymerase. Recently, Mildvan and his coworkers used Mn’+ as a paramagnetic probe to demonstrate that E. coli RNA polymerase ’ This work was supported in part by National Rehas a single tight binding site for the metal search Council of Canada Grant A6938. * To whom correspondence should be sent at the ion cofactor at the catalytic elongation site (10-12). Also, it was determined that there present address:Max-Planck-Institut fur medizinische Forschung, Abteilung Naturstoff Chemie, D-6900 were six weaker binding sites of Mn*+ on Heidelberg, West Germany. the E. coli RNA polymerase molecule but 553
0003-9861/80/100553-12$02.00/O Copyright 0 1980by AcademicPress, Inc. All rights of reproductionin any form reserved.
554
VAISIUS AND HORGEN
the role of these weaker binding sites remains unknown (10). The fact that Mn2+ is not bound at the initiation site (10, 12) does not preclude the possibility of the activating metal ion functioning in the initiation reaction. The requirement for a metal activator at initiation has been clearly demonstrated (13, 14) using an abortive initiation reaction (15) to determine the parameters for the formation of the first phosphodiester bond. As yet, a similar tight binding site for the metal ion activator has not been demonstrated for any eukaryotic RNA polymerase. Perhaps significantly with regard to the complexity which might be expected with eukaryotic RNA polymerases, it was shown that Mn2+ and Mg2+ had distinctly differing effects on the initiation reaction as well as on the elongation reaction of RNA synthesis catalyzed by RNA polymerase I (16). Metal ions are likely to participate in transcription through an interaction with nucleic acids, and it is likely that these interactions will confer variable degrees of specificity on transcription depending on the template and the metal ion. For example, it was reported that RNA polymerase II from calf thymus would form a stable complex with superhelical SV40 DNA form I in the presence of Mn2+ but would not form a similar stable complex in the presence of Mg2+ (1’7). RNA polymerase I from yeast preferentially transcribes ribosomal cistrons with Mg2+ as a cofactor, whereas the same enzyme will randomly transcribe the template with MnZ+ as a cofactor (18). Again, it should be emphasized that little is known regarding the specific effects of metal ions other than Mg2+ or Mn2+ on the association of RNA polymerase with the template despite the fact that many divalent metal ions are known to interact quite differently with a number of reaction sites on nucleic acids (4-8). Since there is little doubt that metal ions play a vital role in the biological process of transcription, it becomes relevant to consider the possible effects of various metal ions on in vitro transcription. There is ample evidence suggesting that many physiologically important metal ions as well as those exhibiting mutagenic effects will accumulate in the cell nucleus (20-25). Because there
was a dearth of information regarding the effects of metal ions other than Mg2+or Mn2+ on in vitro transcription, we have examined the effects of several divalent cations on the activation and inhibition of highly purified RNA polymerases II from the mushroom. A. bisporus and from calf thymus. We expect this information to be useful in future studies of the transcriptional process. MATERIALS
AND METHODS
Chemicals. Calf thymus DNA (Type 1) and nucleoside triphosphates were purchased from Sigma. Tritiumlabeled UTP (37.5 Wmmol) and [3H]CTP (27.8 Ci/ mmol) were purchased from New England Nuclear. Spectroscopically pure manganese chloride and magnesium chloride were purchased from Johnson Matthey Chemicals. All other metals were the highest purity chloride or sulfate salts obtainable from Baker Chemical Company or from Fisher Scientific. Protein detwmin4hms. Protein concentrations were estimated using the Bradford method with prepared reagents from Bio-Rad Laboratories (26). Purijication of RNA polymerase II. All buffers used throughout these experiments were prepared with degassed, deionized water double distilled in glass. RNA polymerase II resistant to a-amanitin from the mushroom A. bisporus was prepared as previously described (27). The purification procedures for RNA polymerase II from calf thymus were similarly modified from Jendrisak and Burgess (28). The purification procedures involes precipitation with polyethylenimine, selective elution of RNA polymerase II from the polyethylenimine precipitate, ammonium sulfate fractionation, batch adsorption and elution with DEAE-cellulose, DEAESephadex chromatography, P-cellulose chromatography, and exclusion chromatography on Bio-Gel agarose, 1.5 M. As shown in Fig. 1, RNA polymerases II from calf thymus and from A. bisporus were purified to near homogeneity as evidenced by polyacrylamide gel electrophoresis under nondenaturing conditions. RNA polymerase assay. The standard assay solution was 75 ~1 having 7% (v/v) glycerol; 75 mM (NH&SO,; 10 mM Tris-HCl (pH 7.9); 0.4 mM each of ATP, CTP, and GTP; 1 &i of [SH]UTP (1 &i/3 nmol); and 5 pg of denatured calf thymus DNA. The concentration of the divalent cation and 2-mercaptoethanol are indicated in the appropriate table or figure legend. In those control assays measuring dependency upon UTP, the labeled substrate was [3H]CTP (1 &i/3 nmol). The concentration and purity of the substrates and template were determined spectroscopically. In all assays without 2mercaptoethanol, the reactions were prepared and carried out under nitrogen. Spectroscopicdeterminations verified that Fe*+ was oxidized in the presence of 2mercaptoethanol, whereas it was not if the reaction so-
EFFECTS
OF DIVALENT
CATIONS
l&ions were incubated under nitrogen in the absence of Z-mercaptoethanol. The enzyme and the template were allowed to preincubate with the metal ions prior to the addition of the substrates. The assay solutions were then incubated for 15 min at 30°C. The reaction was terminated with the addition of 25 ~1 of a solution 16% (w/v) sodium pyrophosphate at 3O”C, and a predetermined aliquot was spotted on a DE-81 (Whatman) disk. The disks were pooled and washed five times for 10 min each time in 0.25 M Na,HP04 (20 ml/disk). The disks were then twice washed in 95% ethanol, and allowed to dry. Radioactivity was counted in a 2:l mixture of PCS (Amersham/ Searle)-xylene scintillation fluid. Corrections were made for the relative counting efficiencies of [“H]UTP and (:‘H]UMP incorporated into RNA as previously described (29). One unit of RNA polymerase activity is defined as the incorporation of 1 nmol of labeled nucleotide into RNA per 15 min at 30°C. Kinetic experiments for Zn 2+inhibition. The effects of Zn2+ on the kinetics of RNA synthesis were determined with a varying substrate concentration. The standard assay conditions were modified as follows: the concentration of ATP, CTP, and GTP were fixed at 0.4 mM with [:jH]CTP (1 pCi/lO nmol) as the labeled nucleotide; the concentration of UTP was varied (0.025, 0.05, 0.075, and 0.10 mM) for each inhibitor concentration and the reactions were incubated for 7.5 min at 25°C. Under these assay conditions, incorporation of the labeled nucleotide always remained less than 2% of the UTP concentration. The effects of Zn*+ on the kinetics of RNA synthesis were determined for varying concentrations of the necessary template cofactor. In these experiments, the standard assay conditions were followed with the exceptions that the DNA concentration was varied ( 1, 2.5, 7.5, 15, and 25 pg/ml) for each inhibitor concentration and that the reactions were incubated for 7.5 min at 25°C. In separate experiments, it was determined that the rate of RNA synthesis was linear for each concentration of DNA used and that the highest concentration used was not saturating. All data were the result of two separate experiments using duplicate assays in each. The best fit lines were determined by use of preprogrammed linear regression analysis.
ON RNA POLYMERASES
555
II
CALFTHYMUS
0
2
6
6
4
Distance ( cm )
A. BISPORUS
P
0.2
I 0.1
2
LAO
2
k
6
6
’
Distance ( cm ) FIG. 1. Polyacrylamide gel electrophoresis under nondenaturing conditions of purified RNA polymerases II from calf thymus and A. bisporus. Electrophoresis was carried out as described by Davis (48). The gels shown are 5% acrylamide and were stained using the method of Blakesley and Boezi (49).
factors in transcription. Reagent grade Mg2+ will serve as a poor activator of RNA polymerase II from A. bisporus whereas ultrapure Mg2+ will ellicit only negligible activity. Using ultrapure Mg2+ with calf thymus RNA polymerase II will cause a decrease in activity but some activity was always measurable under our standard assay conditions. As shown in Table I, all metals tested were done so in the presence and absence of the RESULTS reducing agent 2-mercaptoethanol. This was Several divalent cations were tested as necessary since 2-mercaptoethanol readily activators of transcription catalyzed by RNA complexes with many heavy metals to form polymerase II from calf thymus and A. bis- mercaptides, which could have the apparent porus. As shown in Table I, calf thymus effect of removing metal ions from solution, RNA polymerase II can use Co*+, Fez+, or could facilitate the interaction of free nucleotides with nucleic acids (30). It should Mg2+, and Mn2+as cofactors in transcription. Likewise, RNA polymerase II from A. bis- be noted that highly variable results were porus can use Co2+ as well as Mn2+ as co- obtained with either Cu2+ or Fe2+ as a
556
VAISIUS AND HORGEN TABLE I
THEEFFECTSOFDIVALENTCATIONSASACTIVATORS OFTRANSCRIPTIONBY RNA POLYMERASESIIFROM Agaricus bisporus AND CALF THYMUS~ Specific activity (unitsimg protein) Metal salt (2 mM)
2-Mercaptoethanol (1 mM)
A. bisporus
Calf thymus
BaCl,
+
7.3 0.4
2.0 0
BeSO
+ -
0 0
2.0 0
CdCl,
+ -
0 0
0 0
CaCl,
+ -
0 0
0 0
CoCl,
+ -
49.1 28.6
77.4 74.0
cuso*
+ -
? 0
? 0
FeSO,
+
0 0
? 168.7
MgCl,
+ -
12.5 5.5
23.7 11.3
MnCl, (ultrapure)
+ -
86.2 44.8
125.8 72.9
HgCl,
+ -
NiCl,
+ -
SrCl,
+ -
ZnSO,
+ -
U In those assays without 2-mercaptoethanol, the reactions were carried out under nitrogen.
potential activator if the enzyme was not assayed under nitrogen and in the absence of 2-mercaptoethanol. In Table I, the results obtained for CL?+ or Fez+ in the presence of 2-mercaptoethanol were listed as questionable because the percentage standard deviation for six assays with each metal ion exceeded 10% (normally only values of 5%
or less were tolerated) and control assays for Cu2+ and Fez+ incubated without enzyme were as much as 20 times higher than control assays for the other metals (normal control values were 50-150 cpm). As shown in Fig. 2, titrations of RNA polymerase II from either A. bisporus or calf thymus with Mn2+ exhibit similar activation profiles. There is a slight difference in the concentration of Mn2+ which yields optimal activity with a standardized assay, as the optimal concentration of Mn2+ is 0.8 mM for calf thymus and 1.75 mM for A. bisporus. Quite similar activation profiles are obtained for both enzymes with Co2+as the divalent cation. When calf thymus RNA polymerase II is titrated with Fe2+, a broad activation profile is observed with optimal activity near 4.5 111M.A. bisporus is not even minimally activated by Fe2+ over concentrations ranging from 0.2 to 20 11IM. Tables II and III include several controls which were seen to further substantiate the activation of RNA polymerases II by the metals listed and to exclude the possibility of aberrant catalytic activity with these metals. Assays were run in the absence of a template and in the absence of each of the nucleotides to determine whether or not the metals might catalyze the random polymerization of polyribonucleotides through terminal addition, or catalyze the unprimed synthesis of complementary polyribonucleotides (31, 32). With regard to Fe2+, it was thought that Fe2+ might serve as a reducing agent which very efficiently facilitates the activity of RNA polymerase and the actual activator metal ion might be Mn’+ or another metal ion which remained tightly bound to the enzyme through the isolation procedures. Although this factor cannot be completely dismissed, it seems quite unlikely since the enzyme is not even minimally activated in the absence of an added metal ion over a time course up to 2.5 h. The a-amanitin control further substantiates that the incorporation is catalyzed by RNA polymerases II, and that other metal ion activators do not alter the inhibition by a-amanitin. Because multiple forms of RNA polymerase have yet to be purified from the mushroom and thus their inhibition patterns are unknown, the results of the a-amanitin control are less certain for
EFFECTS OF DIVALENT
CATIONS ON RNA POLYMERASES
557
II
50
25
0 1.0
2.0
3.0
4.0
5.0
6.0
7.0
6.0
9.0
* 0
METAL ION (mM) FIG. 2. (A) The activation of calf thymus RNA polymerase II by divalent metal ions Mn*+ (O), Co2+ (O), M@+(D), and Fe*+ (Cl). (B) The activation of RNA polymerase II from A. bisporus by divalent metal ions Mn2+(O), and Co2+(0). In those assays with Co2+and Fe *+, 2-mercaptoethanol was omitted from the reaction solution and the reactions were carried out under nitrogen.
RNA polymerase II from A. bisporus, which RNA polymerase II. Calf thymus RNA polyis half-maximally inhibited by cw-amanitinat merase II was strongly inhibited by Cu2+ a concentration of 6.5 pg/ml (27), but con- but only at concentrations approaching those tamination with another form of RNA poly- of the metal activator. merase or some other nucleotidyl transferase The inhibition by Mg2+ and the slight inseems unlikely considering the purity of the hibition evidenced with Co2+may not accuenzyme preparations. rately be inhibition but rather a case of As shown in Tables IV and V, several di- these metals functioning as activators but valent cations were shown to be inhibitors with the reaction proceeding much more of RNA polymerase II from the mushroom slowly even in the presence of Mn2+. Also, or calf thymus. Both enzymes were signifi- it can be seen in Tables IV and V that Ba2+ cantly inhibited by Be2+, Cd2+, Hg2+, Ni2+, had an apparent synergistic effect on the and Zn2+ at a concentration lo-fold less than activation of RNA polymerase II from A. bithe optimal concentration of Mn2+which was sporus and calf thymus, and SP exhibited used as the metal activator. Copper (II) and a strong positive synergism on the activity Fe2+were also strong inhibitors ofA. bispom of the mushroom enzyme. As previously
558
VAISIUS TABLE
AND HORGEN
II
REQUISITEASSAYCOMPONENTSFORTHEINCORPORATION OF LABELED NUCLEOTIDE INTO RNA UNDER STANDARDASSAYCONDITIONSBYRNAPOLYMERASE II FROMAgaricus bisporus USING THE OPTIMAL CONCENTRATIONOFEITHERCO~+ORM~~+AS AN ACTIVATOR Specific activity (unitsimg protein) CoCl,
MnCl,
Reaction conditions
(1.5 mM)
(1.75 mM)
Complete standard assay solution - Enzyme -Metal activator -DNA -ATP -CTP -GTP -UTP +a-Amanitin (100 pg/ml)
46.0 0 0 0.07 0.21 0.53 0.49 0.18 6.4
102.5 0 0 0.02 0.26 0.27 0.19 0.09 1.6
inhibition by Zn*+ of RNA polymerases II from both. A. bisporus and calf thymus. Using the above data, the apparent Ki for the inhibition of MnUTP incorporation by Zn*+ (ZnNTP) was calculated to be 0.0027 lllM for RNA polymerase II from A. bisporus and 0.0320 mM for RNA polymerase II from calf thymus. Replots of the data by the Eadie-Hofstee method (35) did not detect any deviation from linearity, thus confirming the observation that inhibition by Zn*+ was apparently competitive. Because the above plots might not clearly differentiate partial competition, Dixon (36) has suggested a method of analysis which would discern partially competitive inhibition. If the reciprocal of the fractional inhibition is plotted against the reciprocal of the inhibitor concentration, the intercept will be unity for pure competitive inhibition whereas the intercept will be greater than unity for partially competitive inhibition (36). Using this method of analysis, the intercepts for curves with each substrate concentration were very near to unity for the experiments using either the mushroom (& = 1.86 + 15%) or the calf thymus (c? = 0.36 ? 21%) enzyme.
stated in a multicomponent reaction such as the RNA polymerase assay, the free metal ion will have a number of potential binding sites, e.g., template, nascent RNA nucleotides, and SO:*. Thus, the apparent synerTABLE III gistic effects of Ba*+ and Sr*+ could result from the displacement of Mn+* from such REQUISITEASSAYCOMPONENTSFORTHEINCORPORAsites where it does not have any direct effect TION OF LABELED NUCLEOTIDE INTO RNA UNDER STANDARDASSAYCONDITIONSBYRNAPOLYMERASE on RNA polymerase. The nature of Zn*+ inhibition on the re- IIFROMCALFTHYMUSUSINGTHEOPTIMALCONCENTRATION OF EITHER Co2+, Fez+, OR MnL+ AS action kinetics of RNA polymerases II were AN ACTIVATOR determined with a varying concentration of the substrate UTP. From calculations using Specific activity the reported stability constants of Mn*+ with (units/mg protein) nucleoside triphosphates (33, 34), it can be reasonably assumed that the true substrates CoCl, MnCI, Reaction FeS04 (4.5 mM) (0.8 mM) (1 mM) are MnNTP3 complexes under the assay conditions conditions employed in this study. It is also quite likely that the true inhibitor is a ZnNTP Complete standard 140.4 68.7 171.6 assay solution complex. Because Mn*+ will bind SOY?with a 0 0 0 -Enzyme KD of 5.0 mM, Zn*+ inhibition might be ex0 0 0 -Metal pected to be dependent upon the SO:’ con- -DNA 0.04 0.05 0.04 centration. As shown in Table VI, Zn*+ -ATP 0.16 0.25 0.09 inhibition increases with an increasing con- -CTP 0.35 0.33 0.29 centration of (NH,),SO,. Graphical analysis -GTP 0.68 0.07 0 of the data by Lineweaver-Burk plots as -UTP 0.12 0.15 0 +cu-Amanitin shown in Fig. 3 demonstrates competitive (0.5 @g/ml) B Abbreviation
used: NTP, nucleoside triphosphate.
2.01
0.51
0.50
EFFECTS OF DIVALENT
CATIONS ON RNA POLYMERASES
Because an inhibitor may also effect the binding or functioning of a cofactor, the effects of Zn2+ inhibition were observed with varying concentrations of the template cofactor. As shown in Fig. 4, Lineweaver-Burk plots indicate an apparent mixed inhibition
THE EFFECTS OF DIVALENT CATIONS AS INHIBITORS BY RNA POLYMERASE OF IN VITRO TRANSCRIPTION II FROM Agaricus bisporus”
559
TABLE V THE EFFECTS
OF DIVALENT
CATIONS
OF IN VITRO TRANSCRIPTION
BY
II FROM CALF
RNA
AS INHIBITORS POLYMERASE
THYMUF
Concentration (mM)
Specific activity (unitslmg protein)
Fractional inhibition
BaCl,
0.1 1.0
132.1 144.5
None None
BeSO*
0.1 1.0
88.4 6.3
0.329 0.952
Metal salt
TABLE IV
II
Concentration (mM)
Specific activity (unitsimg protein)
Fractional inhibition
CdCl,
0.1 1.0
84.2 3.2
0.361 0.976
BaCI,
0.2 2.0
101.6 143.7
None None
CaCl,
0.1 1.0
137.6 116.8
None 0.114
BeSO,
0.2 2.0
32.7 3.4
0.639 0.962
CoClB
0.1 1.0
87.6 78.4
None 0.090
CdCl,
0.2 2.0
11.7 1.4
0.871 0.985
cuso,*
0.1 1.0
81.8 2.9
0.051 0.966
CaCl,
0.2 2.0
90.0 73.4
0.007 0.190
FeSOt
0.1 1.0
104.7 177.9
None None
coc1:
0.2 2.0
42.7 30.5
0.197 0.427
WZ
0.1 1.0
114.7 83.2
0.130 0.369
cuso*4
0.2 2.0
1.2 0.1
0.977 0.999
HgCl;
0.1 1.0
60.5 1.6
0.298 0.981
FeSO<
0.2 2.0
26.3 2.1
0.505 0.960
NiCl,
0.1 1.0
67.4 5.8
0.489 0.956
MgCl,
0.2 2.0
88.8 57.8
0.020 0.362
SrCI,
0.1 1.0
104.7 76.3
0.206 0.421
HgCl$
0.2 2.0
1.3 0
0.976 1.00
ZnSO:
0.1 1.0
5.8 0.7
0.933 0.992
NiCl,
0.2 2.0
60.3 4.8
0.334 0.947
SrCl,
0.2 2.0
100.2 140.2
None None
ZnSO*,
0.2 2.0
1.7 0.1
0.968 0.998
a Under standard assay conditions with 1 mM Mn2+ as the metal activator and 1 mM 2-mercaptoethanol as the reducing agent, the specific activity was 131.8 unitsimg protein. If 2-mercaptoethanol was omitted, the specific activity was 86.2 units/mg protein. An asterisk (*I indicates assays without 2-mercaptoethanol. In those assays without 2-mercaptoethanol, the reactions were carried out under nitrogen.
Metal salt
” Under standard assay conditions with 2 mM Mn2+ as the metal activator and 1 mM 2-mercaptoethanol as the reducing agent, the specific activity was 90.6 units/mg protein. If 2-mercaptoethanol was omitted, the specific activity was 53.2 unitsimg protein. An asterisk (*) indicates the assays without 2-mercaptoethanol. In those assays without 2-mercaptoethanol, the reactions were carried out under nitrogen.
by Zn2+ with respect to the template cofactor for reactions catalyzed by RNA polymerase II from eitherd. bisporus or calfthymus. Zinc as well as other metal ions have been shown to be able to cause the depolymerization of ribonucleic acids (37-39). Although
560
VAISIUS AND HORGEN
merases II. Although the enzymes were isolated from widely divergent species, the THE EFFECTOF(NH,),SO,CONCENTRATION ONTHE RNA polymerases II from the mushroom and INHIBITION WITH 2.5 x lo-” M Zn2+ OF IN VITRO from calf thymus have a similar subunit comTRANSCRIPTION BY RNAPOLYMERASEII position (27, 40), i.e., insofar as can be disFROMCALFTHYMUS" cerned by SDS-polyacrylamide gel electroTABLE VI
WH,),SO, concentration (mM)
Fractional inhibition
0 25 50 75 100 125
0.147 0.158 0.164 0.175 0.227 0.228
(1The fractional inhibition was determined under standard assay conditions with 1 InM Mn*+ as the metal activator.
the reaction conditions are quite different for the depolymerization of RNA by Zn*+ than those used in our assays for RNA polymerase reactions, a control experiment was done to test whether or not the apparent inhibition could be related to depolymerization of the RNA product by Zn2+. The control experiment was as follows: triplicate samples of calf thymus RNA polymerase were assayed under the standard conditions previously described, a second series of samples were assayed as described except that after the alloted incubation period the reactions were terminated with cr-amanitin (10 pg/ml) and then further incubated at the identical time and temperature of the standard reaction, a third series of samples were assayed and terminated by a-amanitin with the simultaneous addition of 0.05 mM Zn*+, and the sampleswere further incubated under standard conditions. There was very good agreement in measured activity for all samples, thus indicating that zinc does not measurably degrade the RNA product under the assay conditions employed in this study. DISCUSSION
Because a comparative study had not previously been done for the divalent cation requirement of eukaryotic RNA polymerases, we have observed the effects of several divalent metal ions on two distinct RNA poly-
FIG. 3. The effect of Zn*+ on the kinetics of RNA synthesis as catalyzed by RNA polymerases II: Lineweaver-Burk plot with varying concentrations of UTP. (A) Lineweaver-Burk plot of the activity of RNA polymerase II from A. bisporus as determined with several concentrations of Zn2+: (W) no Zr?+, (0) 2.5 x lo-'M Zn2+, (0) 2.5 x 10-OM Zn*+, (0) 5.0 x lo-" M Zn'+. The assay conditions are described within the text with the concentration of MnCl, at 1.75 mM, and with the enzyme concentration at either 0.9 or 1.1 pg per reaction. (B) Lineweaver-Burk plot of the activity of RNA polymerase II from calf thymus as determined with several concentrations of Zn*? (a) no ZnZ+, (0)2.5 x 1O-5M Znzf, (0) 5.0 x 10m5 M Zn2+. The assay conditions are described within the text with the concentration of MnCl, at 0.8 mM,and with the enzyme concentration at 0.6 kg per reaction.
EFFECTS
OF DIVALENT
CATIONS
ON RNA POLYMERASES
II
561
[DNA~ pg/mL
1 [OnA]
FIG. 4. The effect of Zn’+ on the kinetics of RNA synthesis as catalyzed by RNA polymerases II: Lineweaver-Burk plot with varying concentrations of the DNA cofactor. (A) Lineweaver-Burk plot of the activity of RNA polymerase II from A. bisporus as determined with several concentrations of Zn2+: (m) no Zn”+, (0) 2.5 x 10-j M Zn2+, (0) 5.0 x 10e5 M Zn2+. The assay conditions are described within the text concentration at either 0.9 or 1.1 pg per reaction. (B) Lineweaver-Burk plot of the activity of RNA polymerase II from calf thymus as determined with several concentrations of Zn2+: (W) no Zn”‘, (0) 5.0 x IO-” M Zn’+, (0) 2.5 x 1O-5 M Zn2+, (0) 5.0 x lo-” M Zn’+. The assay conditions are described within the text with the concentration of MnCl, at 0.8 mM, and with the enzyme concentration at 0.6 pg per reaction.
phoresis. The enzymes share a similar chromatographic behavior on several different ion-exchange resins (l-3, 27, and our unpublished results) and are similar in the general assay conditions yielding optimal activity in vitro (l-3,27). The similarity in catalytic properties has allowed us to use a quite uniform assay for RNA polymerase activity throughout this study. A prominent difference between the two enzymes is the fact that RNA polymerase II from A. bisporus is 3250-fold more resistant to a-amanitin than is calf thymus RNA polymerase II as
determined in our laboratory using identical procedures for each enzyme preparation. A. bisporus was considered an interesting source of RNA polymerase II for these experiments for the additional reason that mushrooms are known to accumulate significant quantities of trace metals (41, 42). Calf thymus RNA polymerase II had the obvious advantage of being one of the best characterized eukaryotic RNA polymerases (l-3). Our results indicate that effects of several divalent cations are generally similar on RNA polymerases II from A. bisporus or
562
VAISIUS
AND
calf thymus. This is particularly evident with Be2+, Cd2+, Hg2+, and Znz+ all of which are strong inhibitors of RNA polymerase II from either calf thymus or A. bisporus. Zinc (II), which is a particularly potent inhibitor of both RNA polymerases, is recognized as an essential growth factor for all organisms (43), and is known to accumulate in the cell nucleus of calf thymus as well as other organisms (21,25). Additionally, nucleotidyl transferases including RNA polymerases have been recognized as zinc metalloenzymes (19,43). Thus, it seems particularly interesting that Zn2+ was shown to be a competitive inhibitor with respect to the substrate (MnUTP). In these experiments both the labeled nucleotide CTP and the nucleotide of varied concentration UTP have not been recognized as initiating nucleotides (44). Although the detailed mechanisms of catalysis are not known for the eukaryotic RNA polymerases, it is generally accepted that there is but a single elongation site and quite possibly a single site for the metal activator at the elongation site. It is very likely that the actual site of Zn2+ competition is the site of the metal activator. Of course, under the technological limitations of our experimental design it is not possible to determine the true free metal concentration or to distinguish between competition with the metal activator site and the nucleotide site if the metal ion is required for the binding of the substrate. Because Zn2+ is a competitive inhibitor with regard to the binding of the substrate, it does not preclude inhibition with respect to the DNA cofactor since the binding of Zn2+ may also alter binding within the ternary complex. The pattern of mixed inhibition, i.e., a competitive as well as a noncompetitive aspect to the inhibition by Zn2+, might be expected considering the complexity of interactions necessary for catalysis. For example, a speculative explanation for mixed inhibition might be that the binding of Zn2+causes a decreased affinity within the ternary complex such that a conformational change is prevented which might be necessary for the proper base pairing between the incoming nucleotide and the template. With such an explanation, competitive inhibition would still be observed with respect to the substrate because increasing the MnNTP con-
HORGEN
centration would decrease the availability of RNA polymerases to the Zn2+ (ZnNTP) inhibitor. Mixed inhibition could also result from the fact that a ZnNTP complex not only competes with substrate binding but also prevents the dissociation of the ternary complex. It should be emphasized that other methods of analyses will be necessary to elucidate the precise nature of the interactions involved in Zn2+ inhibition and to this end it is interesting that the nature of Zn2+ inhibition was similar for the RNA polymerases II from both A. bispoms and calf thymus. The fact that Co2+ and Mn2+ as well as Mg2+ for calf thymus can serve as activating divalent cations might be expected considering that these metals can also activate prokaryotic RNA polymerases as well as other nucleotidyl transferases (19, 45). A striking difference in the RNA polymerases II from calf thymus and A. bisporus is their response to Fe2+. RNA polymerase II from A. bispoms is strongly inhibited by Fe2+, while Fe2+ is the metal ion activator yielding optimal activity for calf thymus RNA polymerase II. These results are interesting in light of the fact that Fe2+/Fe3+is a significant metal ion within the cell nucleus of calf thymus, and in particular with the deoxyribonucleoproteins (20,25,46). The oxidation and reduction of Fe2+/Fe3+ has even been suggested as a possible regulatory mechanism in gene control (7,47). It must be emphasized that free Fe2+/Fe3+ is unlikely to be found in the cell nucleus, and especially unlikely in the concentrations required in this study. Because our experimental design has centered around the use of a quite typical in vitro assay of RNA polymerase, we do not wish to engage in extended speculation on our part attempting to attribute a physiological role for various metal ions with respect to RNA polymerase vis-a-vis the DNA template. However, since there is ample evidence suggesting that many metal ions will accumulate in the cell nucleus (20-25,46), we do think it prudent to consider in future studies the possible role of various metal ions in all aspects of the transcriptional process in eukaryotes. In fact, our preliminary results from a study in progress indicate that the divalent cation activator may influence the
EFFECTS
OF DIVALENT
CATIONS
fidelity of transcription by calf thymus RNA polymerase II on defined templates. In addition, kinetic analysis of transcription with various activating or inhibiting metal ions may prove important in elucidating the chemical properties of the metal ion necessary for RNA synthesis catalyzed by RNA polymerase. ACKNOWLEDGMENTS The authors are particularly thankful for the incisive and helpful criticism of the reviewers. We wish to express our gratitude to Heather MacLeod for her expert technical assistance. We wish to thank Drs. Loeb and Mildvan for sending up preprints of works in press. We are grateful to Drs. Ng and Cummins for helpful discussions during the course of this work. REFERENCES 1. CHAMBON, P. (1974) in The Enzymes (Boyer, P. D., ed.), Vol. 10, pp. 261-331, Academic Press, New York. 2. CHAMBON, P. (1975) An?lu. Rev. Biochem. 44, 613-638. 3. ROEDER, R. (1976) in RNA Polymerase (Losick, R., and Chamberlin, M., eds.), pp. 285-329, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 4. IZATT, R. M., CHRISTENSEN, J. J., AND RYTTING, J. H. (1971) Cheln. Rev. 71, 439-481. WESER, U. (1968) S’truc. Bonding 5, 41-67. EICHHORN, G. L., AND SHIN, Y. A. (1968) J. Amer. Chem. Sot. 90, 7323-7328. SISSOGFF, I., GRISVARD, J., AND GUILL~, E. (1976) Progr. Biophys. Mol. Biol. 31, 165-199. MILDVAN, A. S. (1970) in The Enzymes (Boyer, P. D., ed.), Vol. 2, pp. 445-536. Academic Press, New York. 9. Tu, A. T., AND HELLER, M. J. (1974) in Metal Ions in Biological Systems (Sigel, H., ed.), Vol. 1, pp. l-49, Dekker, New York. 10. KOREN, R., AND MILDVAN, A. S. (1977) Biochemistry 16, 241-249. 11. BEAN, B. L., KOREN, R., AND MILDVAN, A. S. (1977) Biochemistry 16, 3322-3333. 12. STEIN, P. J., AND MILDVAN, A. S. (1978) Biochemistry 17, 2675-2684. 13. SHEMYAKIN, M. F., MALYGIN, A. G., AND PATRUSHEV, L. I. (1978) FEBS Lett. 91, 253-256. 14. MCCLURE, W. R., CECH, C. L., AND JOHNSTON, D. E. (1978) J. Biol. Chem. 253, 8941-8948. 15. JOHNSTON, D. E., AND MCCLURE, W. R. (1976) in RNA Polymerase (Losick, R., and Chamberlin, M., eds.), pp. 413-428, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
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Bio-