Interconvertible Forms of Escherichia coli RNA

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Oct 26, 1970 - dex A-50 column (3 X 25 cm) equilibrated with buffer solution. A. The column was ... acetic acid, and counted for radioactivity ina scintillation.
Proceedings of the National Academy of Sciences Vol. 68, No. 1, pp. 152-154, January 1971

Interconvertible Forms of Escherichia coli RNA Polymerase CESAR A. CHELALA, LUISA HIRSCHBEIN*, AND HECTOR N. TORRESt§ Instituto de Investigaciones Bioquimicas "Fundacion Campomar"; and Facultad de Ciencias Exactas y Naturales, Buenos Aires, Argentina

Communicated by Luis F. Leloir, October 26, 1970 ABSTRACT Escherichia coli RNA polymerase exists in two different forms, one less active than the other under standard assay conditions. Conversion of the active to the inactive form requires ATP-Mg++. The reverse, inactive to active enzyme conversion, requires Mg++. Covalently bound adenylic acid residue(s) are contained in the inactive form.

liquid) against the same solution. The enzyme fractions thus obtained, called 0.25 and 0.40 were stored at 00C. The last fraction was used as source of polymerase activity. Incubation for polymerase conversion was carried out as follows: about 0.140 mg of protein of the 0.40 enzyme fraction, containing the RNA polymerase activity, was incubated at 370C for different periods, with the indicated additions, in a total volume of 0.03 ml. The reaction was stopped by the addition of 1.5 ml of ice-cold buffer solution C. RNA polymerase activity was assayed on 0.15-ml aliquots of these diluted samples plus the following additions: 0.2 mM CTP, UTP, GTP, and (H] ATP (2000 cpm/nmol), 1.6 mM MnCl2, 0.01 mg calf-thymus DNA, 40 mM Tris* HCl buffer, pH 7.4, and 4 mM mercaptoethanol. The total volume was 0.25 ml. Incubations were at 370C for 10 min; the reactions were stopped by the addition of 3 ml of cold 5% trichloroacetic acid. The precipitates were collected on nitrocellulose (Millipore) filters, washed several times with 5% cold trichloroacetic acid, and counted for radioactivity in a scintillation spectrometer using a toluene-2,5 diphenyloxazole-1, 4 bis[2(4-methyl-5-phenyl) oxazolyl]-benzene mixture. In some experiments, the enzyme samples were passed through Sephadex G-25 columns prior to the assay for polymerase activity. The volumes of the 0.40 fraction and the additions were doubled and the reaction was stopped with 0.07 ml of ice-cold buffer solution B. After this, 0.01 ml of a 3% hemoglobin solution was added to the mixture. The samples were then passed through Sephadex G-25 columns (0.8 X 10 cm) equilibrated with buffer solution C. RNA polymerase activity was assayed on the colored effluent as indicated above. The labeling of the enzyme fraction was carried out as follows: 0.14 mg of protein of the 0.40 fraction was incubated with a or -y labeled [32P]ATP (10 mCi/,Mmol) or [U-14C]ATP (0.5 mCi/ymol) with the indicated concentrations of MgCl2. The total volume was 0.03 ml. Incubations were performed at 370C. The reaction was stopped by the addition of 3 ml of cold 5% trichloroacetic acid plus 10 Mmol of ATP. The precipitates were collected and counted for radioactivity as indicated for the polymerase assay. In some experiments, the volumes of the incubation mixtures were increased 60 times and the concentration of MgCl2 was adjusted to 0.74 mM. In these circumstances, the reaction was stopped by the addition of an equal volume of the buffer solution B. After the solution was mixed, the protein was precipitated with 3 volumes of cold saturated ammonium sulfate solution. The precipitate was collected by centrifugation at 10,000 X g for 10 min, then it was taken in a small volume of the same buffer solution. This labeled enzyme fraction was dialyzed (3 changes of liquid) against buffer solution A. The fraction obtained after incubation with [a32P]ATP was chromatographed on DEAE-Sephadex as indicated above,

Transcription of phage and bacterial genes by RNA polymerase seems to be regulated by two different control mechanisms. One of them stimulates the ability of the enzyme to transcribe certain DNA templates, and requires a specific protein factor, called 6 (1). The other control mechanism prevents the transcription by an active polymerase and requires the presence of a specific protein factor-the repressor (2). These two different control mechanisms appear to be quite specific. The first operates as a positive control in the sequential expression of certain genomes; the second exerts a negative control on the genome transcription coordinated with the input of environmental signals. This paper reports that Escherichia coli RNA-polymerase exists in two interconvertible forms of different activity. The results obtained in this work provide evidence for the existence of another mechanism of control of bacterial gene expression. MATERIALS AND METHODS

The following buffer solutions were used: A, 10 mM Tris* HCl (pH 8.4)-i mM EDTA-5 mM ,3-mercaptoethanol; B, 50 mM Tris HCl (pH 7.5)-20 mM EDTA-50 mM NaF-10 mM,mercaptoethanol; C, 50 mM Tris HCl (pH 7.5)-i mM EDTA5 mM NaF-5 mM (3-mercaptoethanol. RNA polymerase was purified from E. coli A-19 by the method of Babinet (3) up to step Si. Column chromatography of this fraction was as follows: about 13 ml of the Si fraction, containing 26 mg protein/ml, was loaded on a DEAE-Sephadex A-50 column (3 X 25 cm) equilibrated with buffer solution A. The column was washed with the same buffer solution until the absorbance of the eluate, measured at 280 nm, was below 0.1. Then it was eluted successively with 250 ml of buffer solution A containing 0.25 M KCl and 250 ml of this buffer solution containing 0.4 M KCl. The fractions from each elution step were pooled and precipitated with solid ammonium sulfate (60 g/100 ml). After centrifugation for 10 min at 10,000 X g, the precipitates were taken up in a minimal volume of buffer solution A, and were dialyzed for 4 hr (2 changes of the *Permanent address: Institut de Microbiologie, Facult6 des Sciences, Orsay, France. $ Career investigator of the Consejo Nacional de Investigaciones Cientificas y Tecnicas (Argentina). § Reprint requests may be addressed to Dr. H. N. Torres, Instituto de Investigaciones Bioqufmicas "Fundaci6n Campomar," Obligado 2490, Buenos Aires (28), Argentina.

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RNA Polymerase of E. coli

except that the elution steps were performed with 3 changes in the concentration of KCl: 0.25, 0.33, and 0.40 respectively. Fractions of 17 ml were collected. On the other hand, the fraction obtained after incubation with [14C ]ATP was loaded on a DEAE-Sephadex column (4.7 X 11 cm) equilibrated with buffer solution A. After the column was washed with 50 ml of the same buffer solution containing 0.25 M KCl, the enzyme was eluted with a linear gradient from 0.25 M to 0.50 M KCl in the same buffer. Fractions of 10 ml were collected; the radioactivity in protein was measured after precipitation of an aliquot of each fraction with 5% trichloroacetic acid and filtration on a nitrocellulose filter. Protein content of the enzyme samples was measured by the method of Lowry et al. (4) Electrophoresis on polyacrylamide gels containing sodium dodecylsulfate was as described elsewhere (5). [a32P]ATP was prepared by enzymatic phosphorylation of labeled 5'-AMP. 5'-['2P]AMP was synthesized by a chemical reaction between isopropylidene adenosine and 32P-labeled inorganic phosphate in the presence of trichloroacetonitrile and triethylamine by a modification of the method of Greenlees and Symons (6). [9y32P]ATP was prepared as previously described (7). RESULTS AND DISCUSSION

Fig. 1 shows that incubation of RNA polymerase preparation in the presence of ATP-Mg++ leads to a time-dependent inactivation of the enzyme. This effect is reversible since the activity was restored by addition of the 0.25 fraction. No important changes in the enzyme activity were observed when the polymerase was incubated without any addition or in the presence of ATP or magnesium ions alone. Some facts indicated that the changes observed in the polymerase activity were the result of interconversions between different molecular entities of the enzyme. These

MI

NUTES

FIG. 1. Inactivation and reactivation of E. coli RNA-polyThe 0.40 enzyme fraction (0.02 ml containing 0.24 mg protein) was incubated in the presence (0,O) or absence (0) of 4.1 mM ATP and 4.1 mM MgCl2. At the time indicated by the arrow, the 0.25 enzyme fraction (5 pl, containing 0.07 mg protein) diluted in 10 mM MgC12 was added to the incubations, performed in the presence of ATP-Mg++ (0). (U), Control reactions containing only the 0.25 enzyme fraction plus ATP-Mg++. merase.

153

TABLE 1. Reaction of labeled ATP with enzyme Radioactivity precipitated with trichloroacetic acid

Conditions

Total Expt. 1 2 3

ATP

(cpm)

IncuMgCl2 bation (mM) (min)

[la82p] [y_32P]

1.6 X 107 5.5 1.4 X 107 5.5 [l4C]aden- 5 X 105 0.74 osine 5.7 X 106 0.74 [a-82p]

(cpm) Incubated

Control

10 6.5

750,000 1200 2,700 900 66,210 1778

6.5

5,000,000 5077

10

Controls received trichloroacetic acid before the incubations began.

changes were observed after a 50-fold dilution of the incubation mixture. Moreover, the same results were obtained when the enzyme samples were passed through Sephadex G-25 columns prior to the assay. In order to determine if an adenylylation or a phosphorylation of the enzyme lead to polymerase inactivation, the 0.40 fraction was incubated with Mg++ and either [a32P]-, [-y32p]_, or [14C ]ATP, precipitated with 5% trichloroacetic acid, filtered, and counted. As shown in Table 1, the incorporation of radioactivity into the trichloroacetic acid-insoluble fraction proceeded only when [a32P]- or [14C JATP was used as substrate. The incorporation from [9y82P]ATP was negligible. Therefore, it appears that inactivation of the polymerase is the result of an adenylylation of the enzyme molecule. In order to verify this presumptive conclusion, the 0.40 fraction was incubated either with [a-32P]- or [14C ]ATP. After that, the enzyme was precipitated with ammonium sulfate, dialyzed, and chromatographed on a DEAE-Sephadex column. As can be seen in Fig. 2 the remaining enzyme activity was eluted in a peak, with almost all of the radioactivity insoluble in 5% trichloroacetic acid. This peak was further precipitated with ammonium sulfate and dialyzed. As shown in Fig. 3, when the labeled enzyme preparation was incubated in the presence of Mg++, it lost radioactivity in a time-dependent reaction. Under these conditions, a 14C-labeled product appeared; it was soluble in 5% trichloroacetic acid. This compound behaved during paper chromatography (ethanol-1 M ammonium acetate, pH 7.4,70:35) as 5'-AMP. Some experimental results indicate that the radioactivity was covalently bound to the protein. The radioactivity coprecipitated with the protein in 5% trichloroacetic acid and, after exhaustive dialysis of the labeled protein against 40% urea and water, the radioactivity remained in the sack. In addition, after tryptic hydrolysis of the labeled enzyme, a radioactive product soluble in 5% trichloroacetic acid was obtained. The evidence thus obtained might indicate that E. coli RNA polymerase interconverts according to the following scheme: ATP-Mg + +(1)

ACTIVE ENZYMEI

Mg + (2)

INACTIVE ENZYME (adenylylated) Some properties of the reactions (1) and (2) responsible for these interconversions were studied.

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Biochemistry: Chelala et al.

Proc. Nat. Acad. Sci. USA

I

0.016 Jo

0

0-

T C0.1

3:

*X

@

0

@

UQ008

20 FRAC TIONS

0

2

4

MINU TE S

6

0

2

6

4

M9gC12

8

10

12

(mM)

(Left) FIG. 2. DEAE-Sephadex column chromatography of the E. coli RNA polymerase labeled with 32P (A) or 14C

(B). (0) Absorb0.15-ml aliquots of the indicated fractions for 20 min at 370C; (A) 32P or 14C, total radioactivity of the indicated fractions precipitable with 5% trichloroacetic acid. Broken line in B indicates the KCl concentration ance

measured at 280

nm;

(0)

enzyme

activity assayed

on

in the elution buffer. (Center) FIG. 3. '4C-labeled enzyme incubated in the presence (-) or absence (0) of 2 mM MgCI2. The labeled enzyme (20 AI, containing 0.018 mg protein) was incubated at 370C for the indicated times. Reactions were stopped with 3 ml of 5% trichloroacetic acid. (Right) FIG. 4. Inactivation of E. coli RNA-polymerase as a function of Mg++ concentration. The 0.40 enzyme fraction was incubated for 5 min (0) in the presence of 4.1 mM ATP; the concentration of MgCl2 was varied. (0), Control, not incubated. The vertical dotted line indicates the concentration of EDTA in the reaction mixture. Other conditions were as in Fig. 1.

Purification of the polymerase on DEAE-Sephadex according to Babinet's method (3), eluting the column in three steps: 0.25, 0.33, and 0.40 M KC1, respectively, led to a polymerase preparation that did not have any ATP-Mg++ effect. On the contrary, when the elution step with 0.33 M KCI was omitted, the enzyme could be inactivated by ATP-Mg++. This result indicates that a factor required for reaction (1) is eluted with 0.33 M KCl. Also, Mn++ could not substitute for Mg++, and GTP, UTP, or CTP could not replace ATP in the inactivation reaction. The extent of the inactivation was studied at a fixed incubation time and constant concentration of ATP, varying the concentration of Mg++. As can be seen in Fig. 4, the maximum rate of inactivation was obtained when the ratio of ATP to Mg++ was about 1. The increase in activity of the polymerase at higher concentrations of Mg++ might indicate an increase in the rate of reaction (2). This assumption is favored by the fact that the liberation of radioactivity from a '4C-labeled enzyme was strongly stimulated in the presence of Mg++ (Fig. 3). This enzyme preparation was submitted to a polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Five protein bands were obtained, with the mobilities of ##r', 6, 1A a, and as were previously described (5, 8). Probably one of these bands corresponds to the enzyme responsible for reaction (2). In the experiment shown in Fig. 1, the addition of a protein fraction eluted from the DEAE-Sephadex column with 0.25 M KCI (0.25 fraction) almost reverses the effect of ATP-Mg++ in a time-dependent reaction. As this fraction contains an active adenylate kinase and 5'-nucleotidase, its effect could be due to the removal of ATP. Since the observed effects would be a balance between reactions (1) and (2), it is obvious that a w

diminution in the rate of reaction (1) leads to an increase in the polymerase activity. No polymerase activity was detected in the 0.25 fraction. In conclusion, our results show that E. coli RNA polymerase can exist in two different forms, one more active than the other under the conditions of the assay. The less-active form appears to be the adenylylated form of the active enzyme. Interconversion between active and inactive forms of E. coli RNA polymerase may play an important role in the transcription of bacterial genes during the cell cycle of growth. This kind of control mechanism differs from those previously described (1, 2). We express our special gratitude to Dr. Jos6 M. Olavarria for his invaluable assistance, to Dr. Luis F. Leloir for his helpful criticism, and to other members of the Instituto de Investigaciones Bioqufmicas for their collaboration. This work was supported in part by grants from the U.S. Public Health Service (GM 03442) and the Consejo Nacional de Investigaciones Cientfficas y Tecnicas (Argentina). 1. Travers, A. A., and R. R. Burgess, Nature, 222, 537 (1969). 2. Jacob, F., and J. Monod, J. Mol. Biol., 3, 318 (1961). 3. Babinet, Ch., Biochem. Biophys. Res. Commun., 26, 639 (1967). 4. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 5. Hirschbein, L., J. M. Dubert, and Ch. Babinet, FEBS Lett., 3, 260 (1969). 6. Greenlees, A. W., and R. H. Symons, Biochim. Biophys. Acta, 119, 241 (1966). 7. Torres, H. N., and C. A. Chelala, Biochim. Biophys. Acta, 198, 495 (1970). 8. Zillig, W., E. Fuchs, P. Palm, D. Rabussay, and K. Zechel, in RNA Polymerase and Transcription, ed. L. G. Silvestry (North Holland, Amsterdam, 1969), p. 151.