[64] Cyanobacterial DNA-dependent RNA polymerase

PHYSIOLOGY AND METABOLISM

592

[64]

[64] C y a n o b a c t e r i a l D N A - D e p e n d e n t R N A P o l y m e r a s e By GEORGE BORBELY and GEORGEJ. SCHNEIDER Introduction Cyanobacterial gene expression requires the transfer of genetic information from DNA to RNA molecules which is mediated by DNA-dependent RNA polymerase (EC 2.7.7.6). The RNA polymerases purified from cyanobacterial sources are similar to bacterial RNA polymerases in the following respects: divalent metal ion requirement for enzymatic activity, rifamycin sensitivity, a-amanitin insensitivity, and requirement for a DNA template. The enzyme catalyzes the initiation, elongation, and termination of polyribonucleotide chains using ribonucleoside triphosphates as substrates. Since RNA polymerases of cyanobacterial origin have a number of properties which are common to other prokaryotic RNA polymerases, purification steps and strategies established for Escherichia coli and/or other enteric bacteria are useful guides in purification of the enzyme. 1,2 The role and functions of cyanobacterial RNA polymerases and the mechanism by which the enzyme functions require a great deal of further investigation. Here we describe the purification of Anabaena sp. PCC 7120 DNA-dependent RNA polymerase. 3 Assay Method The R N A polymerase assay mixture contains, in a final volume of 35 /zl,the following: 50 m M Tris-HCl, p H 8.0 at 25 °, 10 m M MgCI2, 2.5 m M 2-mercaptoethanol, 0.5 m M each of UTP, GTP, and CTP, 0.125 m M [8-3H]ATP (0.5-1.0/zCi of tritiatedA T P diluted to a specific radioactivity of 0.06/zCi/nmol), and 0. I mg/ml chicken erythrocyte D N A . D N A synthesis is initiated by adding enzyme extract (5-10/zg enzyme protein) to the assay mixture. After 10 min at 37 ° the reaction mixture is spotted onto W h a t m a n G F / C filterswhich are washed batchwise 3 times for 5 rain in 5 % (w/v) trichloroacetic acid. After drying under a heat lamp, the filters are counted in toluene base scintillation fluid by standard procedures. iR. R. Burgess, in " R N A Polymerase" (R. Losick and M. Chambcrlin,eds.),p. 69. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1976. 2 M. J. Chamberlin, in " R N A Polyrnerase" (R. Losick and M. Chamberlin, eds.),p. 20. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1976. 3 G. J. Schneider,N. E. Turner,C. Richaud,G. Borbely,and R. Hasclkorn,J. Biol. Chem. 262, 14633(1987). METHODS IN ENZYMOLOGY, VOL. 167

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

[64]

CYANOBACTERIALRNA POLYMERASE

593

One unit of RNA polymerase activity is defined as 1 nmol of [3H]ATP incorporated in 10 rain at 37°. Specific activity is defined as units/mg protein. In the early phase of enzyme purification, the use of volume activity units (nmoles of radioactive substrate incorporated by 1 ml enzyme fraction in 10 rain at 37°) more conveniently follows the RNA polymerase activity. In addition to measurement of enzymatic activity by incorporation of radioactive substrates into acid-precipitable material, the purified cyanobacterial RNA polymerase should be analyzed for subunit structure by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis.4 Template DNAs such as calf thymus or chicken erythrocyte DNA, cyanophage AS-1 or N-1 DNA (obtained by standard purification steps and phenol extraction), and the dAT copolymer are prepared in l0 mM Tris-HCl, pH 8.0, 10 mM NaC], 0.1 mM EDTA solution, and usually 20, 5, or 2/zg are added per assay, respectively. The assay mixture is prepared by mixing the ingredients just prior to use from more concentrated solutions containing the salts, buffer, unlabeled nucleoside triphosphates, DNA, labeled substrate, and RNase-free double-distilled H 2 0 . Comments on the Assay

Beside [3H]ATP, 14C-labeled ATP or UTP (0.1-1 mCi/mmol) can be used as alternative substrates for the measurement of RNA polymerase activity (theoretically, any one of the nucleoside triphosphates may be used as the label). Control experiments using UTP as a labeled substrate and employing rifampicin (10/zg/ml) are recommended to ensure that the incorporation is the result of DNA-dependent RNA polymerase activity and not another contaminating and copurified enzyme(s) in the extract. For further discussion and review, see Burgess: Burgess and Jendrisak, 6 and Chamberlin and Ryan. 7 Purification Procedure The procedure described is for the isolation of the RNA polymerase holoenzyme and core polymerase from Anabaena sp. PCC 7120 (ATCC 27893). Cell disruption is accomplished by French pressure cell treatment. After ultracentrifugation of the lysate, RNA polymerase is further 4 U. K. Laemmli, Nature (London) 227, 680 (1970). 5 R. R. Burgess, J. Biol. Chem. 244, 6160 (1969). 6 R. R. Burgess and J. J. Jendrisak, Biochemistry 14, 4634 0975). 7 M. Chamberlin and T. Ryan, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 15, p. 87. Academic Press, New York, 1982.

594

PHYSIOLOGY AND METABOLISM

[64]

purified by Polymin P (polyethyleneimine, Sigma) precipitation. Final purification involves gel filtration chromatography (BioGel A-1.5 m, BioRad) and binding to Heparin-Sepharose (Pharmacia). The holoenzyme and core R N A polymerase are separated on a BioRex 70 (Bio-Rad) column. The or factor is purified by SDS-gel electrophoresis. 8 Buffers. All buffers and solutions are prepared from double-distilled water and the highest grade chemicals available. Buffer A: 20 m M Tris-HCl, pH 8.0, 10 m M MgC12, 50 m M KCI, 0.1 m M EDTA, 0.1 m M dithiothreitol, 1.0 mM phenylmethylsulfonyl fluoride, 10% (v/v) glycerol Buffer G: 20 m M Tris-HCl, pH 8.0, 10 m M MgC12, 50 m M KC1, 0.1 mM EDTA, 0.1 m M dithiothreitol, 1.0 mM phenylmethylsulfonyl fluoride Polymin P: 10% solution of polyethyleneimine titrated with 12 N HC1 to pH 8.0 and stored at 4 ° Storage Buffer: Buffer A solution with 50% (v/v) glycerol Saturated ammonium sulfate: Enzyme grade ammonium sulfate is used to prepare a saturated solution at 25 ° in water and the pH then adjusted to 7.5-8.0 with K O H Growth of Cyanobacterial Cells. Anabaena sp. filaments are grown in Kratz and Myers' medium 9 with combined nitrogen under axenic conditions to mid exponential phase of growth. Illuminated 10- to 15-liter carboys are stirred with magnetic stirrers and aerated with sterile air containing 1% CO2. Yields are 1.5-2.0 g (wet weight) cells per liter. (Growth of Anabaena sp. in Allen medium l° or BG-11 ii works equally well for RNA polymerase purification.)

Purification Steps The cells are harvested with a Sorvall continuous flow system (SzentGy6rgyi-Blum apparatus) in an SS-34 rotor at 4 ° and washed in Buffer G. The packed cell pellet is suspended in 2 volumes of Buffer A and then passed through a cold French pressure cell twice at 20,000 psi. The cell lysate is ultracentrifuged for 90 min at 120,000 g. The RNA polymerase is precipitated from this supernatant by slow addition with stirring in 10% polyethyleneimine solution to a final concentration of 0.05%. After centrifugation (12,000 g for 10 min at 4°C in a 8 D. A. Hager and R. R. Burgess, Anal. Biochem. 109, 76 (1980). 9 W. A. Kratz and J. Myers, Am. J. Bot. 42, 282 (1955). l0 M. M. Allen, J. Phycol. 4, 1 (1968). H R. Rippka, J. Deruelles, J. B. Waterbury, M. Herdman, and R. Y. Startler, J. Gen. Microbiol. U l , 1 (1979).

[64]

CYANOBACTERIALRNA POLYMERASE

595

Sorvall SS-34 rotor), the pellet is extracted with 25 ml of Buffer A containing 400 mM KC1 by resuspension with a Teflon homogenizer. After centrifugation (as above) the washed pellet is dissolved in I0 ml Buffer A containing 2.0 M KCI and the polyethyleneimine removed by 3 ammonium sulfate precipitations; each time the pellet is resuspended in 2.0 M KCI-Buffer A and 2.3 volumes of saturated ammonium sulfate solution added. The final pellet is resuspended in 5 ml Buffer A containing 400 mM KCI and dialyzed against the same buffer. Gel Filtration on BioGel A-1.5 m. The dialyzed extract is loaded on a 2.8 x 83 cm column of BioGel A-1.5 m equilibrated with Buffer A containing 400 m M KCI and then eluted at a flow rate of 15 ml/hr with the same buffer. The active enzyme fractions are pooled and precipitated by adding solid ammonium sulfate to 60% saturation. The precipitate, after centrifugation, is resuspended and dialyzed against Buffer A containing 200 mM KCI. Heparin-Sepharose Chromatography. The dialyzed protein fraction from the previous step is applied to a 1.5 x 7 cm Heparin-Sepharose column equilibrated with Buffer A containing 200 mM KC1. After washing the column with 2 volumes of Buffer A containing 200 mM KC1, the RNA polymerase is step-eluted with Buffer A containing 300 mM KC1. The pure enzyme is precipitated with 60% saturated ammonium sulfate as above and resuspended and dialyzed against storage buffer (containing 50 mM KC1 and 50% glycerol) for storage at - 2 0 °. BioRex 70 Chromatography for Core and Holoenzyme. Three milligrams of RNA polymerase from the Heparin-Sepharose column is separated into core- and holoenzyme using a 1 × 6 cm freshly prepared BioRex 70 column equilibrated with Buffer A containing 50 mM KC1. The column is washed with 2 column volumes of this buffer, and the core enzyme is step-eluted with Buffer A containing 200 mM KC1. The holoenzyme elutes from the BioRex 70 column with 500 mM KC1 in Buffer A. The purification of Anabaena sp. RNA polymerase from 16 g wet weight of cells is summarized in Table I. A total of 2.7 mg of enzyme protein is obtained; the overall yield of 46% is based on the amount of/3 subunit analyzed by Coomassie blue-stained SDS-polyacrylamide gels. 7 Protein samples from each step of the purification are compared in Fig. 1.

Purification of Anabaena sp. RNA Polymerase o- Factor Since it has not been possible to obtain the RNA polymerase o- subunit chromatographically, the o- subunit is purified from 1.25 mg of BioRex 70 holoenzyme pool separated into component subunits on SDS-polyacrylamide gels. The o- subunit is isolated and renatured as described. 8 The

596

PHYSIOLOGY AND METABOLISM

[64]

TABLE I PURIFICATION OF Anabaena RNA POLYMERASE Purification

Volume

Total

step

(ml)

protein (mg)"

High-speed

78

733

Total units t' 854

Specific activity (U/mg) 1.2

Yield (%)' 100

supernatant PE! eluate BioGel pool HeparinSepharose pool

6.2 3.65

42 "6.1

1061 483

25 79

95 66

1.08

2.7

422

156

46

" Measured by Bio-Rad protein assay with bovine serum albumin as standard. t' One unit equals incorporation of 1 nmol [3H]ATP in 10 min at 37°. ' Calculated by quanitation of/3 subunit on Coomassie-stained SDS gels.

electrophoretically purified o- factor fully reconstitutes the activity of holoenzyme when added to core in a run-off transcription assay using a DNA fragment containing a T4 bacteriophage early promoter as a template12; the transcripts are analyzed on a 5% polyacrylamide sequencing gel (Fig. 2). Comments on the Purification Steps. The Polymin P precipitation may be performed on a French pressure cell-treated crude extract and afterward extracted with high-salt-containing Buffer A. 7 As an additional step DEAE-cellulose (DE-52, Whatman) chromatography can be helpful in the purification of RNA polymerase; the enzyme activity is step eluted with Buffer A containing 250 mM KCI. Properties of Cyanobacterial RNA Polymerases. Thus far, DNA-dependent RNA polymerase has been purified and partially characterized from Anacystis nidulans (Synechococcus sp.), 13-17,20 Fremyella diplosiphon, 18 Anabaena cylindrica, 19 and Anabaena 3p. PCC 7120. 3 RNA ~2M. Chamberlin, R. Kingston, M. Gilman, J. Wiggs, and A. DeVera, this series, Vol. 101, p. 540. 23 K. van der Helm and W. Zillig, Hoppe-Seyler's Z. Physiol. Chem. 341t, 302 (1967). 24 K. van der Helm and W. Zillig, FEBS Lett. 3, 76 (1969). 15 F. Herzfeld and W. Zillig, Fur. J. Biochem. 24, 242 (1971). 26 F. Herzfeld and N. Rath, Biochim. Biophys. Acta 274, 431 (1974). 17.F. Herzfeld and M. Kiper, Eur. J. Biochem. 62, 189 (1976). 28 S. S. Miller and L. Bogorad, Plant Physiol. 62, 995 (1978). 19 G. Borbely and N. G. Carr, unpublished observations. 20 M. Kuman6, N. Tomioka, K. Shinozaki, and M. Sugiura, Mol. Gen. Genet. 202, 173 (1986).

1

2

3

4

-13'

"--(3

---(~

FIG. 1. Silver-stained gel of Anabaena RNA polymerase purification fractions. Samples from each step of the purification were separated on a 12.5% SDS-polyacrylamide gel and stained with silver. Lanes: 1, 9.4 ~g high-speed supematant; 2, 6.7/xg PEI eluate; 3, 1.7/xg BioGel A-1.5 m pool; 4, 1.25/xg Heparin-Sepharose pool. Subunit assignments are at right.

598

PHYSIOLOGY AND METABOLISM

A

[64]

B

1

2

3

1

2

3

4

5

6

7

8

_

RI

a

-

FIG. 2. Sigma subunit identification and holoenzyme reconstitution. (A) Silver-stained gel of(lane 1) 2.6/~g holoenzyme, (2) 4.0/zg core enzyme separated on BioRex 70, (3) 0.2 tzg gel-purified o- subunit with bovine serum albumin. (B) Run-off transcription assays using a bacteriophage T4 early promoter, analyzed on a 5% acrylamide sequencing gel. Lanes: 1, 1.3/zg holoenzyme; 2, 0.2/~g core enzyme; 3, 0.2/zg o- subunit; 4-8, 0.2/zg core enzyme plus (4) 0.01 /zg o-, (5) 0.02/~g o-, (6) 0.05/~g o-, (7) 0.1 /~g o-, or (8) 0.2/zg or.

polymerases of different origin exhibit slight differences for divalent metal ion requirement. The Anacystis nidulans and Anabaena sp. PCC 7120 enzymes require Mg 2+ (10-20 mM) for maximal activity; Mn 2+ can partially replace Mg 2+ for enzyme activity. Anabaena cylindrica enzyme requires Mn ~÷ (2-4 mM) for optimal activity, and Mg 2÷ shows only a limited ability to replace Mn ~÷. In the case of the Fremyella diplosiphon enzyme, Mg 2÷ (30 raM) or Mn 2+ (5 mM) works equally well for maximal activity, but the enzyme shows an extreme instability in the presence of salts.

[65]

EXTRACELLULARPROTEINS

599

TABLE II SUBUNIT MOLECULAR WEIGHTS OF VARIOUS CYANOBACTERIAL R N A POLYMERASES

Subunit a Source

[3'

[3

~t

O"

0£

Anabaena sp. PCC 7120 Anabaena cylindrica Anacystis nidulans Fremyella diplosiphon

171 185 190 161

124 125 145 134

66 47 72 72

52

41 30 38 41

Xb

93 91

Ref.

3 19 16 18

a Subunits are given by their sizes on SDS gels in kilodaltons. b Unrelated to Anabaena sp. PCC 7120 subunits.

Anabaena 7120 RNA polymerase has, at 66 kDa, an additional subunit as part of the core enzyme. This subunit copurifies in a 1 : 1 : 1 : 2 ratio with the other core subunits fl',/3, and a (named by analogy to the E. coli RNA polymerase subunits). It has been proposed that this subunit be named the 3~ subunit) On the basis of published copurification and stoichiometric data, the 47-kDa subunit from Anabaena cylindrica 19 and the 72-kDa subunits from Anacystis nidulans 16 and Fremyella diplosiphon 18 have been assigned as the corresponding 3' subunits. The 93-kDa Anabaena cylindrica and the 91-kDa Fremyella diplosiphon subunits, although close to the apparent size of E. coli ~ when analyzed by S D S polyacrylamide gel electrophoresis, do not have a counterpart in the Anabaena sp. PCC 7120 enzyme. Table II summarizes the apparent molecular sizes of the subunits of cyanobacterial RNA polymerases, as established by SDS-polyacrylamide gel electrophoresis.

[65] E x t r a c e l l u l a r P r o t e i n s

By D. J. SCANLAN and N. G. CARR Introduction

Cyanobacteria have been widely reported to excrete peptides and proteins into their culture media. In spite of their apparent quantitative importance little is known regarding the composition, and less about the function of extracellular proteins and peptides, some aspects of which are discussed below. It is, of course, established that some of the potent cyanobacterial toxins are peptides, but there is no clear evidence that METHODS IN ENZYMOLOGY, VOL. 167

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.