Cell,
Vol.
67, 155-167,
October
4, 1991,
Copyright
0 1991
by Cell
Press
The Phosphorylated Form of the Enhancer-Binding Protein NTRC Has an ATPase Activity That Is Essential for Activation of Transcription David S. Weiss, Jacques Batut,” Karl E. Klose, John Keener,t and Sydney Kustu Department of Plant Pathology and Department of Molecular and Cell Biology University of California Berkeley, California 94720
Summary The NTRC protein of enteric bacteria is an enhancerbinding protein that activates transcription in response to limitation of combined nitrogen. NTRC activates transcription by catalyzing formation of open complexes by RNA polymerase (9 holoenzyme form) in an ATP-dependent reaction. To catalyze open complex formation, NTRC must be phosphorylated. We show that phosphorylated NTRC has an ATPase activity, and we present biochemical and genetic evidence that NTRC must hydrolyze ATP to catalyze open complex formation. It is likely that all activators of 054 holoenzyme have an ATPase activity. Introduction In addition to the most abundant sigma factor, cr70,eubacteria employ alternative sigma factors that confer different promoter specificities on the core form of RNA polymerase. Whereas most alternative sigma factors allow transcription of genes whose products contribute to a common physiological response, ds4 differs in that it is needed for transcription of genes whose products have diverse physiological roles (for reviews see Kustu et al., 1989; Thony and Hennecke, 1989). c? is not homologous to other known sigma factors, and a54-dependent promoters are characterized by an unusual spacing of the recognition elements (-12, -24). o”-dependent genes appear to share a mechanism of transcriptional activation-transcription by os4 holoenzyme depends on an activator protein bound to sites located at a distance from the transcriptional start. The proteins that activate transcripiion by os4 holoenzyme have in common a domain of ~240 amino acid residues that appears to be directly responsible for transcriptional activation (Figure 1A). Moreover, all of the activators contain a recognizable nucleotide-binding motif within this domain (Ronson et al., 1987). The best-studied activator of o” holoenzyme is the nitrogen-regulatory protein NTRC of enteric bacteria (also called NRI). The mechanism of activation by NTRC has been studied intensively at the major promoter for the g/nA gene, which encodes glutamine synthetase. To activate
‘Present address: Laboratolre de Biologie Plantes-Microorganismes, CNRS-INRA, net-Tolosan, Cedex, France. fPresent address: Department of Biological California, Irvine, California 92717.
Moleculaire BP27, Chemistry,
des Relations F31326 CastaUniversity
of
transcription, NTRC binds to enhancer-like sites (Reitzer and Magasanik, 1986) located over 100 bases upstream of the start siteof transcription and contacts the polymerasepromoter complex directly by means of DNA loop formation (Reitzer et al., 1989; Su et al., 1990; Wedel et al., 1990). NTRC activates transcription by catalyzing the isomerization of closed recognition complexes between a54 holoenzyme and theg/nA promoterto open complexes, in which the DNA strands are locally denatured around the transcriptional start site (Sasse-Dwight and Gralla, 1988; Popham et al., 1989). The isomerization reaction requires ATP (Popham et al., 1989). Control of transcription at css4-dependent promoters appears to be accomplished primarily by modulating the function of the various activator proteins. For different activator proteins, function is modulated by different mechanisms and in response to different physiological signals (Kustu et al., 1989; Thony and Hennecke, 1989). The activity of NTRC is controlled positively by phosphorylation by NTRB (also called NRII) (Ninfa and Magasanik, 1986; Keener and Kustu, 1988; Weiss and Magasanik, 1988); phosphorylation increases under conditions of nitrogen limitation. The phosphorylation site in NTRC is within an amino-terminal domain of -120 residues (Figure 1A) (Keener and Kustu, 1988). A homolog of this domain has been found on more than 20 bacterial regulatory proteins whose activity is either known or thought to be modulated by phosphorylation (for reviews see Albright et al., 1989; Stock et al., 1989); these are the receiver proteins of “twocomponent” regulatory systems, which play a prominent role in signal transduction in eubacteria (Nixon et al., 1986; Kofoid and Parkinson, 1988). Little is known about the mechanism by which phosphorylation regulates the function of receiver proteins. Although phosphorylation of NTRC is not required for binding to its enhancer-like sites in the g/nA promoter regulatory region, it is absolutely required for its function as a transcriptional activator. We show that NTRC has an ATPase activity that is essential for open complex formation. We also show that a primary role for phosphorylation of NTRC is to stimulate its ATPase activity. We infer that other activators of 05” holoenzyme must also have an ATPase activity but that their ability to hydrolyze ATP will not be regulated by phosphorylation in every case. Results Phosphorylation of NTRC Activates an ATPase Activity We have previously demonstrated that ATP is required directly for the formation of open complexes by crw holoenzyme at the glnA promoter (Popham et al., 1989) as well as being required for phosphorylation of NTRC. To show a direct ATP requirement, we used a mutant form of NTRC (NTRCSIGOF[constitutive]) that has weak ability to activate transcription without being phosphorylated. (The activity of this mutant form, which has a lesion in its central domain
Cell 156
ATP
o0 Figure
1. Domain
structure
ADP
10
20 30 Time (mid
40
50
! 10
' 7 20 Time (mf$
40
1 so
of NTRC
(A) NTRC from S. typhimurium is 469 amino acids long and contains domains for regulation (-120 amino acids), transcriptional activahon (~240 amino acids), and DNA binding (-60 amino acids) (reviewed in Weiss et al., 1991). The regulatory domain is phosphorylated at aspartate 54 (circled “P”). The activation domain contains an ATPbinding motif (GESGSGK in one-letter amino acid code). The underlined glycine in the ATP-binding motif is at position 173 and is mutated to asparagine in one of the NTRCY’““‘“’ proteins (see below). The amino acids that are identical in all activators of os4 holoenzyme whose sequence is known are indicated in black. (6) Location of amino acid substitutions in an NTRCc”“s”‘““” protein (above) and several NTRC’“P’“““O’ proteins (below). Note that all repressor mutations occur in the transcriptional activation domain. With one exception they change residues that are identical in all of the activators whose sequence has been determined. (The exception: glutamate 208 is aspartate in FHLA.)
B
1
OJ 0 Figure
[Serl60-*Phe; Figure 1 B], is greatly increased by phosphorylation [Popham et al., 1989; Weglenski et al., 19891.) When studied in the absence of its cognate phosphotransferase NTRB, NTRCS’GOF still required ATP to catalyze open complex formation; ADP and analogs of ATP with nonhydrolyzable 8-r bonds failed to substitute (Popham et al., 1989). Since NTRC has a sequence motif found in many nucleotide-binding proteins, we hypothesized that NTRC might have to hydrolyze ATP to catalyze open complex formation. Consistent with this hypothesis, we have found that both wild-type NTRC and NTRCSIGOF(see below) have ATPase activity. For the wild-type protein this activity is dependent upon phosphorylation. We assayed the ATPase activity of NTRC-phosphate by quantitating release of inorganic phosphate, P,, under conditions in which NTRC-phosphate was generated in situ-in reaction mixtures containing NTRC, NTRB, and [yJ2P]ATP. We generated NTRC-phosphate in situ, rather than using purified material, because NTRC-phosphate has a rapid autophosphatase activity that makes it difficult to purify in high yield or maintain in constant amount during experiments (Figure 2A) (Keener and Kustu, 1988; Weiss and Magasanik, 1988). To detect ATPase activity during a phosphorylation reaction, it was necessary to distinguish P, released by the ATPase from P, released by the autophosphatase. In our first experiments we monitored P, release at early time points during a phosphorylation reaction-when the level of NTRC-phosphate was low-to minimize the contribution of the autophosphatase to the observed P, produc-
3 2
2. Time
Course
of Phosphorylation
of NTRC
(A) NTRC (1 hM) was phosphorylated by NTRB (100 nM) at an ATP concentration of 0.25 mM, as described in Experimental Procedures (see also Keener and Kustu, 1988). The time courseof phosphorylation (filled diamonds) was obtained by initiating a reaction with labeled MgATP (3360 cpmlpmol). After 20 min the autodephosphorylation rate was determined by adding a portion of the reaction mixture to prewarmed unlabeled MgATP to give a final ATP concentration of 13 mM (open circles). The half-life of NTRC-phosphate determined from these data is 3.2 min. The inset diagrams the phosphorylation and autophosphatase activities that underlie the time course of phosphorylation. (B) In the same experiment shown in (A), a second reaction (open diamonds) was initiated with unlabeled ATP at 0.25 mM and allowed to proceed for 10 min before addition of carrier-free [y-32P]ATP (final specific activity 3085 cpmlpmol). Samples were taken at the times indicated to monitor the rate at which the label equilibrated into NTRCphosphate.
tion. Control reactions showed that wild-type NTRC alone neither incorporated nor released much P, (Table 1A and see Table 3). Similarly, NTRB alone did not release much P, (Table 1A; NTRB is a phosphotransferase and incorporates P, under these conditions). In contrast, when NTRC and NTRB were present together, there was a high initial rate of P, incorporation, which is known to be largely into NTRC, and a higher rate of P, release (Table 1B). Since the rate of P, release exceeded the rate of incorporation, it could not be accounted for solely by the autophosphatase activity of NTRC-phosphate. (Recall that NTRC-phosphate is a$cumulating during the initial phase of the reaction [Figure 2A], so the rate of phosphorylation must exceed the rate of autodephosphorylation.) We hypothesized that the high initial rate of P, release was due, at least in part, to an ATPase activity of NTRC-phosphate.
Phosphorylated 157
Table
1. ATPase
Experiment
Proteins Conditions
A
NTRC NTRB
B
C
NTRC
Activity
Is an ATPase
of Phosphorylated
and
Rate of P, Incorporation (pmollminll0
NTRC
~1)
Rate of P, Release (pmoll min/lO ~1)
95% pure as judged-by silver staining of SDS-polyacrylamide gels. ATPase Assay A standard ATPase
assay
contained
50 mM Tris-acetate
(pH i3.0), 40
Phosphorylated 165
NTRC
Is an ATPase
mM potassium chloride, 5.4 mM magnesium chloride, 0.1 mM EDTA, 3% glycerol, 1 mM DTT, and 0.1 mglml acetylated BSA. Concentrated ATP was made equimolar with MgCI, and was then diluted to 4 mM. Carrier-free [y-32P]ATP (>5000 Cilmmol; Amersham or New England Nuclear) was diluted to 3.0 nCi/fu with water. After addition of NTRC and NTRBtothe buffer(final concentrations 1 mM and 100 nM, respectively), the mixture was warmed for 4 min at 37X, and the reaction was then initiated by adding .I vol of the stock MgATP. After allowing 10 min for the concentration of NTRC-phosphate to reach steady state, .I vol of the diluted [y-32P]ATP was added (final specific activity -2000 cpmlpmol). After 5 min, a IO nl sample for measuring P, was withdrawn and added to 990 ul of ice-cold 1 N formic acid. At 15, 20, and 25 min after addition of the label, IO nl samples for measuring NTRCphosphate were withdrawn and spotted onto Whatman ET31 filters (2 cm square), which were washed with trichloroacetic acid (TCA) and scintillation-counted as previously described (Corbin and Reimann, 1974; Keener and Kustu, 1988). The steady-state level of NTRCphosphate reported is the average of these three measurements. For some experiments (Table 3 and Figure 5) we used an acetatebased buffer that is closer in composition to our transcription buffer and that stimulated ATPase activity. This buffer is 75 mM Tris-acetate (pH 8.0) 100 mM potassium acetate, 25 mM potassium chloride, 27 mM ammonium acetate, 8 mM magnesium acetate, 0.025 mM EDTA, 2.5% glycerol, 1.5 mM DTT, and 0.1 mglml acetylated BSA. Free P, was separated from nucleoside mono-, di-, and triphosphates on polyethyleneimine (PEI) cellulose plates (Brinkman or J. T. Baker) by ascending chromatography using 0.4 M K,HPO,, 0.7 M boric acid (Bochner and Ames, 1982). Running the plates first in water improved performance. The identity of P, was determined by cochromatography with 32P, (Amersham). Samples of 5 nl were spotted onto chromatography plates, which were air dried, soaked in methanol for 5 min, air dried again, and then run. After chromatography the plates were air dried and autoradiographed. The film was used as a guide to cut out the phosphate spots from the plastic-backed TLC plates for counting radioactivity. Values reported for P, release (and NTRCphosphate) were corrected for background by subtracting the corresponding values from mock reactions that contained no proteins. To assay for hydrolysis of a-labeled nucleoside triphosphates, the following modifications were made. The labeled nucleotide was added in .l vol to achieve a specific activity of about 200 cpmlpmol of ATP. At the times indicated, 10 nl samples for determination of nucleoside diphosphate (NDP) were withdrawn and added to 90 ul of 1 .l N formic acid containing 4 mM EDTA and the corresponding NTP and NDP at 4 mM each as carriers. NDP was separated from nucleoside mono-and triphosphates by ascending chromatography on PEI cellulose plates using 0.5 M LiCI, 1 M formate (Richet and Raibaud, 1989). The identity of labeled nucleotides was determined by cochromatography with standards and detection with shortwave UV light using PEI plates with a UV,,, fluorescent background (Brinkman). To assay for hydrolysis of ATPTS, NTRCs’MFwas prephosphorylated with unlabeled ATP for 10 min in the acetate-based ATPase buffer. Then .I vol of [y-32P]ATP or [y-%]ATPyS (New England Nuclear) was added. At 5, 30, and 90 min after addition of label, 10 nl samples were withdrawn and added to 990 nl of formate that contained 1 mM cold thiophosphate as carrier and chromatographed as for P,. The identity of thiophosphate was determined by cochromatography and detection with Ellman’s reagent (Sigma; Deakin et al., 1963). Hydrolysis was quantitated by determining the ratio of P, or thiophosphate to ATP or ATPyS using a phosphorimager (Molecular Dynamics). This mode of analysis compensated for pipetting errors. To assay for inhibition of ATP hydrolysis by ATPyS or ADP, NTRCSIMF was prephosphorylated with unlabeled ATP for 10 min. Aliquots of the reaction were then transferred to a series of tubes, each of which contained .1 vol of [y-3ZP]ATP and enough ATPyS or ADP to achieve the final concentrations indicated in Figure 7C. Samples for determrning P, release were withdrawn 1 min after addition of label. Assay for Open Complex Formation Open complex formation was quantitated by means transcription assay(Popham et al., 1989; Wedel et al., template was the supercoiled plasmid pJES528. This construction and characterization wrll be described tains a wild-type g/nA promoter regulatory region that
of a single-cycle 1990). The DNA plasmid, whose elsewhere, condirects syntheses
of a 155 nucleotide transcript containing no uridine residues. Omission of uridine triphosphate (UTP) during transcription allowed formation of the expected 155 nucleotide transcript and prevented synthesis of other transcripts. To assay inhibition of open complex formation by ATPyS or ADP, reactions were performed with components at the following final concentrations: 0.1 mglml acetylated BSA, 30 nM core RNA polymerase from E. coli, 50 nM as4 from S. typhimurium, 100 nM NTRCSIQF protein (constitutive), and 1 nM plasmid template. Components were added to buffer at 4% in the order indicated above. Mixtures were then warmed for 5 min at 37”C, and reactions were initiated by adding ATP, with or without ATPyS or ADP, to achieve a final ATP concentration of 1 mM in a total volume of 25 ~1. The final concentrations of ATPyS and ADP are indicated in Figure 7A. After 5 min, formation of open complexes was terminated by addition of heparin to a final concentration of 0.1 mglml. After an additional 5 min, guanosine triphosphate (GTP, final concentration 0.4 mM) and cytidine triphosphate (CTP, final concentration 0.1 mM; it contained 5 FCi of [@P]CTP [New England Nuclear]) were added to allow synthesis of transcripts. After 5 min, transcripts were precipitated, isolated by gel electrophoresis, and quantified as described. Radioactivity in transcript bands was converted to femtomole of transcript; the specific activity of transcripts was determined by multiplying the specific activity of CTP by64, the number of cytidine residues per transcript. To titrate ATP in the presence of a fixed concentration of ATPyS the following two modifications were made: formation of open complexes was initiated by adding a mix of ATP and ATPyS to achieve the concentrations indicated in Figure 78, and ATP was added to at least 0.1 mM (in addition to GTP and CTP) during synthesis of transcripts. Acknowledgments D. S. W. and J. B. contributed equally to this work and should be considered first authors. We thank Andy Wedel for assistance with transcriptionexperimentsandthephosphorimager, SuePorterfor help with the mobility shift assay, Anne Moon for some of the cloning, and Richard Burgess for the gift of core RNA polymerase. We are grateful to Mike Chamberlin, Caroline Kane, Jon Goldberg, Elizabeth Greene, and members of our laboratory for helpful discussions. J. B. was supported by a grant from lnstitut National de la Recherche Agronomique, France. This work was supported by Public Health Service grant GM38361 from the National Institutes of Health to S. K. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenl’ in accordance with 18 USC Section 1734 solely to indicate this fact. Received
June
20, 1991
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Vol.
67, 155-167,
October
4, 1991,
Copyright
0 1991
by Cell
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The Phosphorylated Form of the Enhancer-Binding Protein NTRC Has an ATPase Activity That Is Essential for Activation of Transcription David S. Weiss, Jacques Batut,” Karl E. Klose, John Keener,t and Sydney Kustu Department of Plant Pathology and Department of Molecular and Cell Biology University of California Berkeley, California 94720
Summary The NTRC protein of enteric bacteria is an enhancerbinding protein that activates transcription in response to limitation of combined nitrogen. NTRC activates transcription by catalyzing formation of open complexes by RNA polymerase (9 holoenzyme form) in an ATP-dependent reaction. To catalyze open complex formation, NTRC must be phosphorylated. We show that phosphorylated NTRC has an ATPase activity, and we present biochemical and genetic evidence that NTRC must hydrolyze ATP to catalyze open complex formation. It is likely that all activators of 054 holoenzyme have an ATPase activity. Introduction In addition to the most abundant sigma factor, cr70,eubacteria employ alternative sigma factors that confer different promoter specificities on the core form of RNA polymerase. Whereas most alternative sigma factors allow transcription of genes whose products contribute to a common physiological response, ds4 differs in that it is needed for transcription of genes whose products have diverse physiological roles (for reviews see Kustu et al., 1989; Thony and Hennecke, 1989). c? is not homologous to other known sigma factors, and a54-dependent promoters are characterized by an unusual spacing of the recognition elements (-12, -24). o”-dependent genes appear to share a mechanism of transcriptional activation-transcription by os4 holoenzyme depends on an activator protein bound to sites located at a distance from the transcriptional start. The proteins that activate transcripiion by os4 holoenzyme have in common a domain of ~240 amino acid residues that appears to be directly responsible for transcriptional activation (Figure 1A). Moreover, all of the activators contain a recognizable nucleotide-binding motif within this domain (Ronson et al., 1987). The best-studied activator of o” holoenzyme is the nitrogen-regulatory protein NTRC of enteric bacteria (also called NRI). The mechanism of activation by NTRC has been studied intensively at the major promoter for the g/nA gene, which encodes glutamine synthetase. To activate
‘Present address: Laboratolre de Biologie Plantes-Microorganismes, CNRS-INRA, net-Tolosan, Cedex, France. fPresent address: Department of Biological California, Irvine, California 92717.
Moleculaire BP27, Chemistry,
des Relations F31326 CastaUniversity
of
transcription, NTRC binds to enhancer-like sites (Reitzer and Magasanik, 1986) located over 100 bases upstream of the start siteof transcription and contacts the polymerasepromoter complex directly by means of DNA loop formation (Reitzer et al., 1989; Su et al., 1990; Wedel et al., 1990). NTRC activates transcription by catalyzing the isomerization of closed recognition complexes between a54 holoenzyme and theg/nA promoterto open complexes, in which the DNA strands are locally denatured around the transcriptional start site (Sasse-Dwight and Gralla, 1988; Popham et al., 1989). The isomerization reaction requires ATP (Popham et al., 1989). Control of transcription at css4-dependent promoters appears to be accomplished primarily by modulating the function of the various activator proteins. For different activator proteins, function is modulated by different mechanisms and in response to different physiological signals (Kustu et al., 1989; Thony and Hennecke, 1989). The activity of NTRC is controlled positively by phosphorylation by NTRB (also called NRII) (Ninfa and Magasanik, 1986; Keener and Kustu, 1988; Weiss and Magasanik, 1988); phosphorylation increases under conditions of nitrogen limitation. The phosphorylation site in NTRC is within an amino-terminal domain of -120 residues (Figure 1A) (Keener and Kustu, 1988). A homolog of this domain has been found on more than 20 bacterial regulatory proteins whose activity is either known or thought to be modulated by phosphorylation (for reviews see Albright et al., 1989; Stock et al., 1989); these are the receiver proteins of “twocomponent” regulatory systems, which play a prominent role in signal transduction in eubacteria (Nixon et al., 1986; Kofoid and Parkinson, 1988). Little is known about the mechanism by which phosphorylation regulates the function of receiver proteins. Although phosphorylation of NTRC is not required for binding to its enhancer-like sites in the g/nA promoter regulatory region, it is absolutely required for its function as a transcriptional activator. We show that NTRC has an ATPase activity that is essential for open complex formation. We also show that a primary role for phosphorylation of NTRC is to stimulate its ATPase activity. We infer that other activators of 05” holoenzyme must also have an ATPase activity but that their ability to hydrolyze ATP will not be regulated by phosphorylation in every case. Results Phosphorylation of NTRC Activates an ATPase Activity We have previously demonstrated that ATP is required directly for the formation of open complexes by crw holoenzyme at the glnA promoter (Popham et al., 1989) as well as being required for phosphorylation of NTRC. To show a direct ATP requirement, we used a mutant form of NTRC (NTRCSIGOF[constitutive]) that has weak ability to activate transcription without being phosphorylated. (The activity of this mutant form, which has a lesion in its central domain
Cell 156
ATP
o0 Figure
1. Domain
structure
ADP
10
20 30 Time (mid
40
50
! 10
' 7 20 Time (mf$
40
1 so
of NTRC
(A) NTRC from S. typhimurium is 469 amino acids long and contains domains for regulation (-120 amino acids), transcriptional activahon (~240 amino acids), and DNA binding (-60 amino acids) (reviewed in Weiss et al., 1991). The regulatory domain is phosphorylated at aspartate 54 (circled “P”). The activation domain contains an ATPbinding motif (GESGSGK in one-letter amino acid code). The underlined glycine in the ATP-binding motif is at position 173 and is mutated to asparagine in one of the NTRCY’““‘“’ proteins (see below). The amino acids that are identical in all activators of os4 holoenzyme whose sequence is known are indicated in black. (6) Location of amino acid substitutions in an NTRCc”“s”‘““” protein (above) and several NTRC’“P’“““O’ proteins (below). Note that all repressor mutations occur in the transcriptional activation domain. With one exception they change residues that are identical in all of the activators whose sequence has been determined. (The exception: glutamate 208 is aspartate in FHLA.)
B
1
OJ 0 Figure
[Serl60-*Phe; Figure 1 B], is greatly increased by phosphorylation [Popham et al., 1989; Weglenski et al., 19891.) When studied in the absence of its cognate phosphotransferase NTRB, NTRCS’GOF still required ATP to catalyze open complex formation; ADP and analogs of ATP with nonhydrolyzable 8-r bonds failed to substitute (Popham et al., 1989). Since NTRC has a sequence motif found in many nucleotide-binding proteins, we hypothesized that NTRC might have to hydrolyze ATP to catalyze open complex formation. Consistent with this hypothesis, we have found that both wild-type NTRC and NTRCSIGOF(see below) have ATPase activity. For the wild-type protein this activity is dependent upon phosphorylation. We assayed the ATPase activity of NTRC-phosphate by quantitating release of inorganic phosphate, P,, under conditions in which NTRC-phosphate was generated in situ-in reaction mixtures containing NTRC, NTRB, and [yJ2P]ATP. We generated NTRC-phosphate in situ, rather than using purified material, because NTRC-phosphate has a rapid autophosphatase activity that makes it difficult to purify in high yield or maintain in constant amount during experiments (Figure 2A) (Keener and Kustu, 1988; Weiss and Magasanik, 1988). To detect ATPase activity during a phosphorylation reaction, it was necessary to distinguish P, released by the ATPase from P, released by the autophosphatase. In our first experiments we monitored P, release at early time points during a phosphorylation reaction-when the level of NTRC-phosphate was low-to minimize the contribution of the autophosphatase to the observed P, produc-
3 2
2. Time
Course
of Phosphorylation
of NTRC
(A) NTRC (1 hM) was phosphorylated by NTRB (100 nM) at an ATP concentration of 0.25 mM, as described in Experimental Procedures (see also Keener and Kustu, 1988). The time courseof phosphorylation (filled diamonds) was obtained by initiating a reaction with labeled MgATP (3360 cpmlpmol). After 20 min the autodephosphorylation rate was determined by adding a portion of the reaction mixture to prewarmed unlabeled MgATP to give a final ATP concentration of 13 mM (open circles). The half-life of NTRC-phosphate determined from these data is 3.2 min. The inset diagrams the phosphorylation and autophosphatase activities that underlie the time course of phosphorylation. (B) In the same experiment shown in (A), a second reaction (open diamonds) was initiated with unlabeled ATP at 0.25 mM and allowed to proceed for 10 min before addition of carrier-free [y-32P]ATP (final specific activity 3085 cpmlpmol). Samples were taken at the times indicated to monitor the rate at which the label equilibrated into NTRCphosphate.
tion. Control reactions showed that wild-type NTRC alone neither incorporated nor released much P, (Table 1A and see Table 3). Similarly, NTRB alone did not release much P, (Table 1A; NTRB is a phosphotransferase and incorporates P, under these conditions). In contrast, when NTRC and NTRB were present together, there was a high initial rate of P, incorporation, which is known to be largely into NTRC, and a higher rate of P, release (Table 1B). Since the rate of P, release exceeded the rate of incorporation, it could not be accounted for solely by the autophosphatase activity of NTRC-phosphate. (Recall that NTRC-phosphate is a$cumulating during the initial phase of the reaction [Figure 2A], so the rate of phosphorylation must exceed the rate of autodephosphorylation.) We hypothesized that the high initial rate of P, release was due, at least in part, to an ATPase activity of NTRC-phosphate.
Phosphorylated 157
Table
1. ATPase
Experiment
Proteins Conditions
A
NTRC NTRB
B
C
NTRC
Activity
Is an ATPase
of Phosphorylated
and
Rate of P, Incorporation (pmollminll0
NTRC
~1)
Rate of P, Release (pmoll min/lO ~1)
95% pure as judged-by silver staining of SDS-polyacrylamide gels. ATPase Assay A standard ATPase
assay
contained
50 mM Tris-acetate
(pH i3.0), 40
Phosphorylated 165
NTRC
Is an ATPase
mM potassium chloride, 5.4 mM magnesium chloride, 0.1 mM EDTA, 3% glycerol, 1 mM DTT, and 0.1 mglml acetylated BSA. Concentrated ATP was made equimolar with MgCI, and was then diluted to 4 mM. Carrier-free [y-32P]ATP (>5000 Cilmmol; Amersham or New England Nuclear) was diluted to 3.0 nCi/fu with water. After addition of NTRC and NTRBtothe buffer(final concentrations 1 mM and 100 nM, respectively), the mixture was warmed for 4 min at 37X, and the reaction was then initiated by adding .I vol of the stock MgATP. After allowing 10 min for the concentration of NTRC-phosphate to reach steady state, .I vol of the diluted [y-32P]ATP was added (final specific activity -2000 cpmlpmol). After 5 min, a IO nl sample for measuring P, was withdrawn and added to 990 ul of ice-cold 1 N formic acid. At 15, 20, and 25 min after addition of the label, IO nl samples for measuring NTRCphosphate were withdrawn and spotted onto Whatman ET31 filters (2 cm square), which were washed with trichloroacetic acid (TCA) and scintillation-counted as previously described (Corbin and Reimann, 1974; Keener and Kustu, 1988). The steady-state level of NTRCphosphate reported is the average of these three measurements. For some experiments (Table 3 and Figure 5) we used an acetatebased buffer that is closer in composition to our transcription buffer and that stimulated ATPase activity. This buffer is 75 mM Tris-acetate (pH 8.0) 100 mM potassium acetate, 25 mM potassium chloride, 27 mM ammonium acetate, 8 mM magnesium acetate, 0.025 mM EDTA, 2.5% glycerol, 1.5 mM DTT, and 0.1 mglml acetylated BSA. Free P, was separated from nucleoside mono-, di-, and triphosphates on polyethyleneimine (PEI) cellulose plates (Brinkman or J. T. Baker) by ascending chromatography using 0.4 M K,HPO,, 0.7 M boric acid (Bochner and Ames, 1982). Running the plates first in water improved performance. The identity of P, was determined by cochromatography with 32P, (Amersham). Samples of 5 nl were spotted onto chromatography plates, which were air dried, soaked in methanol for 5 min, air dried again, and then run. After chromatography the plates were air dried and autoradiographed. The film was used as a guide to cut out the phosphate spots from the plastic-backed TLC plates for counting radioactivity. Values reported for P, release (and NTRCphosphate) were corrected for background by subtracting the corresponding values from mock reactions that contained no proteins. To assay for hydrolysis of a-labeled nucleoside triphosphates, the following modifications were made. The labeled nucleotide was added in .l vol to achieve a specific activity of about 200 cpmlpmol of ATP. At the times indicated, 10 nl samples for determination of nucleoside diphosphate (NDP) were withdrawn and added to 90 ul of 1 .l N formic acid containing 4 mM EDTA and the corresponding NTP and NDP at 4 mM each as carriers. NDP was separated from nucleoside mono-and triphosphates by ascending chromatography on PEI cellulose plates using 0.5 M LiCI, 1 M formate (Richet and Raibaud, 1989). The identity of labeled nucleotides was determined by cochromatography with standards and detection with shortwave UV light using PEI plates with a UV,,, fluorescent background (Brinkman). To assay for hydrolysis of ATPTS, NTRCs’MFwas prephosphorylated with unlabeled ATP for 10 min in the acetate-based ATPase buffer. Then .I vol of [y-32P]ATP or [y-%]ATPyS (New England Nuclear) was added. At 5, 30, and 90 min after addition of label, 10 nl samples were withdrawn and added to 990 nl of formate that contained 1 mM cold thiophosphate as carrier and chromatographed as for P,. The identity of thiophosphate was determined by cochromatography and detection with Ellman’s reagent (Sigma; Deakin et al., 1963). Hydrolysis was quantitated by determining the ratio of P, or thiophosphate to ATP or ATPyS using a phosphorimager (Molecular Dynamics). This mode of analysis compensated for pipetting errors. To assay for inhibition of ATP hydrolysis by ATPyS or ADP, NTRCSIMF was prephosphorylated with unlabeled ATP for 10 min. Aliquots of the reaction were then transferred to a series of tubes, each of which contained .1 vol of [y-3ZP]ATP and enough ATPyS or ADP to achieve the final concentrations indicated in Figure 7C. Samples for determrning P, release were withdrawn 1 min after addition of label. Assay for Open Complex Formation Open complex formation was quantitated by means transcription assay(Popham et al., 1989; Wedel et al., template was the supercoiled plasmid pJES528. This construction and characterization wrll be described tains a wild-type g/nA promoter regulatory region that
of a single-cycle 1990). The DNA plasmid, whose elsewhere, condirects syntheses
of a 155 nucleotide transcript containing no uridine residues. Omission of uridine triphosphate (UTP) during transcription allowed formation of the expected 155 nucleotide transcript and prevented synthesis of other transcripts. To assay inhibition of open complex formation by ATPyS or ADP, reactions were performed with components at the following final concentrations: 0.1 mglml acetylated BSA, 30 nM core RNA polymerase from E. coli, 50 nM as4 from S. typhimurium, 100 nM NTRCSIQF protein (constitutive), and 1 nM plasmid template. Components were added to buffer at 4% in the order indicated above. Mixtures were then warmed for 5 min at 37”C, and reactions were initiated by adding ATP, with or without ATPyS or ADP, to achieve a final ATP concentration of 1 mM in a total volume of 25 ~1. The final concentrations of ATPyS and ADP are indicated in Figure 7A. After 5 min, formation of open complexes was terminated by addition of heparin to a final concentration of 0.1 mglml. After an additional 5 min, guanosine triphosphate (GTP, final concentration 0.4 mM) and cytidine triphosphate (CTP, final concentration 0.1 mM; it contained 5 FCi of [@P]CTP [New England Nuclear]) were added to allow synthesis of transcripts. After 5 min, transcripts were precipitated, isolated by gel electrophoresis, and quantified as described. Radioactivity in transcript bands was converted to femtomole of transcript; the specific activity of transcripts was determined by multiplying the specific activity of CTP by64, the number of cytidine residues per transcript. To titrate ATP in the presence of a fixed concentration of ATPyS the following two modifications were made: formation of open complexes was initiated by adding a mix of ATP and ATPyS to achieve the concentrations indicated in Figure 78, and ATP was added to at least 0.1 mM (in addition to GTP and CTP) during synthesis of transcripts. Acknowledgments D. S. W. and J. B. contributed equally to this work and should be considered first authors. We thank Andy Wedel for assistance with transcriptionexperimentsandthephosphorimager, SuePorterfor help with the mobility shift assay, Anne Moon for some of the cloning, and Richard Burgess for the gift of core RNA polymerase. We are grateful to Mike Chamberlin, Caroline Kane, Jon Goldberg, Elizabeth Greene, and members of our laboratory for helpful discussions. J. B. was supported by a grant from lnstitut National de la Recherche Agronomique, France. This work was supported by Public Health Service grant GM38361 from the National Institutes of Health to S. K. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenl’ in accordance with 18 USC Section 1734 solely to indicate this fact. Received
June
20, 1991
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