Cro Regulatory Protein Specified by Bacteriophage X

STRUCTURE, DNA-BINDING, AND REPRESSION OF RNA SYNTHESIS*

(Received for publication, February 1, 1977)

YOSHINORI TAKEDA, ATIS FOLKMANIS,$ AND HARRISONECHOLS

From the Department of Molecular Biology, University of California, Berkeley, California 94720

The Cro protein specified by bacteriophage A is a repressor
of the genes expressed early in phage development and is
required for a normal late stage of lytic growth. We have
purified Cro protein to virtual homogeneity and analyzed its
structure and properties as a DNA-binding protein and re-
pressor of RNA synthesis. To confirm that the protein is the
binding activity, and establishes the capacity of Cro to func-
tion as a specific repressor of RNA synthesis initiated at the
early promoter sites of A-DNA. Our biochemical results indi-
cate that the physiological differences between c1 and Cro may
be attributable to the different binding capacities of the two
proteins.

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product of the cro gene, we have also shown that a missense
EXPERIMENTAL PROCEDURES
mutation in the ero gene leads to a product that is more
temperature- and salt-sensitive in its DNA-binding property. Materials
As purified, Cro protein is a dimer of identical subunits of Nucleic Acids - Bacteriophage DNA was prepared from purified
molecular weight 8600. The purified protein binds to A-DNA phage by phenol extraction as described previously (10, 12).
carrying the specific binding sites (operators oL and oli) with “Chicken blood” DNA and Escherichia coli tRNA were obtained from
Calbiochem.
an estimated dissociation constant of lo-‘” M to lo-” M; there Proteins-E. coli RNA polymerase was prepared as described by
is also weaker binding to other sites on DNA, as found for Burgess and Jendrisak (13). Termination factorp, purified according
other DNA-binding regulatory proteins. In a purified tran- to Roberts (141, was the gift of J. Galluppi and J. Richardson,
scription system, the Cro protein is an effective and specific University of Indiana. Pancreatic DNase was obtained from Worth-
ington and ovalbumin, chymotrypsin, myoglobin, and bovine serum
repressor of RNA synthesis from the N and cro genes; thus albumin from Schwarz/Mann.
Cro is an autorepressor which regulates its own synthesis. A Bacterial and Phage Strains - The bacterial host used to prepare
comparison of the properties of the two A repressor proteins, Cro protein was CGOOSu- . The infecting phage for large scale prepa-
CI and Cro, indicates that cI is a “strong repressor” special- rations of wild type Cro protein was hNam53ulu3cIaml4Sam7; to
ized for complete turnoff of lytic functions needed for the characterize the temperature-sensitive mutant protein, parallel in-
fections by hNam53ulu3cro+ and hNam53Num7ulo3cro~ were used, in
maintenance of lysogeny, whereas Cro is a “weak repressor” which the cro- mutation was tof2 (15). The A mutations and the
specialized for a gradual turnoff of early viral genes that rationale for using them have been described in more detail previ-
potentiates the late stage of lytic development. ously (12). In brief, N- mutation eliminates production of most h
proteins besides Cro, ulu3 may increase Cro production, cI- mutation
eliminates the DNA-binding activity of c1 protein, and S- mutation
prevents cell lysis.
The temperate bacteriophage A specifies two repressor pro- Other Mate&Es- Cellulose (CFll) and phosphocellulose (PII)
teins, c1 and Cro, which carry out regulatory functions essen- were obtained from Whatman and Sephadex G-75 (140 to 120 CL
particle size) from Pharmacia. h-DNA-cellulose was prepared as
tial for different aspects of the viral life cycle. The c1 protein described by Alberts and Herrick (16). Ultrapure ammonium sulfate
acts under conditions of stable lysogeny to maintain repression was obtained from Schwarz/Mann, acrylamide and sodium dodecyl
of the integrated viral DNA; Cro protein acts during lytic sulfate from Bio-Rad, unlabeled nucleoside triphosphates from
development to turn off the expression of the phage genes Sigma, [5-“HIUTP from Radiochemical Center, Amersham, I IY-
active early after infection (l-6) and thus potentiates the 82P]UTP from New England Nuclear, and rifampicin from Lepetit.
early-late switch in expression of viral genes. Methods
The c1 protein has been purified and extensively character- DNA-binding Assay for Cro Protein- The standard assay was
ized in vitro for binding to specific operator sites on X-DNA and done essentially as described previously (10, 12). The assay measures
ability to repress RNA synthesis initiated at the X promoter the retention of A-I”‘P]DNA on a nitrocellulose filter by virtue of its
sites active early during viral development (7-11) (Fig. 1). In a tight binding to Cro protein; for purification an excess (loo-fold) of
unlabeled “chicken blood” DNA was added to compete for the bind-
previous paper, we presented data indicating that Cro protein
ing of proteins that associate with DNA but lack specificity for A-
is a DNA-binding protein which binds to the same operator DNA. To further differentlate the operator-specific Cro protein from
region of A-DNA as does c1 (12). This report describes the other h-DNA-binding proteins, we did parallel assays with h-DNA
purification of Cro protein to apparent homogeneity, presents and Aimm434-DNA (which is the same as A-DNA except for the
a more detailed characterization of its structure and DNA- operator-containing immunity region). The standard assay mixture
contained 0.2 pg of A-I,L’PIDNA and 20 pg of chicken blood DNA in
* This research was supported in part by Grant GM 17078 from the 0.1 ml of “binding buffer”: 10 rnM Tris/HCl (pH 7.31, 20 mM KCl, 10
National Institute of General Medical Sciences. rnM MgCl,, 0.2 rnM dithiothreitol, and 0.2 rnM EDTA. After incuba-
$ Postdoctoral Fellow of the National Cancer Institute. tion for 10 min at O”, the mixture was filtered with a nitrocellulose

6177
6178 Cro Protein of Phage X
TABLE I

12s ‘IS
22
Purification of Cro protein
22
Recomb.aenes CHIT cm cII-Redn.aenes Q Volume Total protein Spz$tcyac- Yield
M i-4
Fraction
1 )
PR unitsing %
PL ml w protein
OL OR
CrO PM I. Crude extract 550 4455 -a -0
8
II. Phosphocellulose 50 39.5 157 100
FIG. 1. The activity of c1 and Cro inferred from experiments in III. Sephadex G-75 40 5.2 415 35
uiuo and in vitro. To maintain the repression essential for lysogeny, 6 0.88 3355 48
IV. DNA-cellulose 1
the cI protein acts at operators oL and oH to repress leftward and
V. DNA-cellulose 2 4 0.60 4000 39
rightward transcription initiated at the early promoter sites pL and
pR, respectively; the c1 protein also activates leftward transcription 0 The binding is not specific for A-DNA in the crude extract, and so
of the c1 gene initiated at the maintenance promoter phi The repres- no value for activity can be given at this stage.
sion activity of c1 keeps the entire phage genome repressed because
theN gene product is required as a positive regulator of other genes.
The Cro protein acts to turn off (or turn down) the expression of early sodium dodecyl sulfate as described by Weber and Osborn (19).
genes during the late stage of lytic development through its capacity Velocity sedimentation was performed in 10 to 30% glycerol gra-
to act at oL and os to repress early gene transcription; the Cro protein dients containing 10 mM Tris/HCl (pH 7.3), 0.2 mM EDTA, 0.2 mM
probably represses transcription of the c1 gene initiated at phi Other dithiothreitol, and KC1 as noted; sedimentation was at 49,000 rpm
genes and genetic regions on the figure are: ~11 and cIII, genes which for 24 h in a Spinco SW 50.1 rotor. Amino acid analysis of Cro protein
activate initial expression of the c1 gene during infection; Q, gene for was carried out with a Beckman analyzer after a 20-h hydrolysis of
protein that activates late gene transcription; Repin, replication; the protein in 6 N HCl at 110” in an evacuated tube; analysis for
Recomb, recombination. The waved lines above the A genome indi- tryptophan was done spectrophotometrically by the method of
cate early RNAs observed in uiuo and in vitro; the S values indicate Beaven and Holiday (20). Protein was determined by the method of
approximate sedimentation coefficient of the RNAs. Lowry et al. (21) with bovine serum albumin as a standard. Salt
concentrations in gradient fractions were measured with a conduc-

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tivity meter (Radiometer type CDM2e).
filter (B6, Schleicher and Schuell), washed with 0.5 ml of binding
buffer, dried, and the retained radioactivity was determined by
RESULTS
liquid scintillation counting. For assays involving purified Cro pro-
tein, the chicken blood DNA was omitted. One unit of DNA-binding Purification of Cro Protein
activity is defined as the quantity sufficient to retain 1 pg of A-DNA
on the filter. The binding values are corrected for a “background” of A summary of the purification of Cro protein is presented in
DNA that is retained on the filter in the absence of binding proteins; Table I. Unless otherwise noted, all operations were performed
this varies from 5 to 15% with DNA preparation. For the experi- at O-4”.
ments to determine the kinetic and equilibrium binding constants
(Figs. 7 to 9), the washing procedure was modified by the use of a 0.5- Growth of Znfected Cells-A culture (100 liters) of Esche-
ml wash with 45% ethanol. This procedure gave higher and more richia coli CGOOSu- was grown in a New Brunswick Fermen-
reproducible binding data with lower levels of Cro protein, presum- ter at 30” in a broth containing 2% Difco Bacto-tryptcme, 1%
ably because dissociation during the washing step did not occur (no yeast extract, 0.5% NaCl, and 0.2% maltose. When the A,!,,, of
decrease in binding was found up to at least 1 ml of wash volume).
Binding values similar to those of the ethanol wash were obtained by
the culture reached 1.0, MgCl, was added to 10 mM and h
reducing the volume of the standard washing buffer (0.1 ml), but the phage (strain Nam53cIam14ulv3Sum7) was added at a multi-
data were erratic with a higher level of background binding. plicity of 10 phages/cell. After 60 min at 30”, the cells were
RNA Synthesis and Analysis-RNA synthesis was carried out harvested by centrifugation in a Sharples continuous flow
using purified RNA polymerase and hb2- or Ab2imm434-DNA in a
reaction in which RNA chains were initiated in the presence of
centrifuge, resuspended in 50 ml of 10% sucrose, 50 mM Tris/
rifampicin; the use of DNA carrying the b2 deletion and of rifampicin HCI (pH 7.8, quick-frozen, and stored at -20”. The procedure
results in a larger fraction of RNA from the promoter sites on A-DNA yielded 150 g of cells.
used in viva for early RNA (10, 14, 17). The standard reaction Preparation of Extract-The frozen cells were thawed and
mixture (0.1 ml) contained: 100 rnM Tris/HCl (pH 7.2), 40 mM NaCl, mixed with 80 ml of 2 mg/ml of lysozyme in 250 mM Tris/HCl
10 mM MgCIZ, 1 rnM ATP, GTP, CTP, and 0.1 mM [5JH]- or [(Y-
32PlUTP, 1 pg of rifampicin, 0.7 pg of RNA polymerase, and 3 pg of (pH 7.51, 1 mM EDTA, and 19 ml of 4 M NaCl, 10 mM 2-
A-DNA. The A-DNA and Cro protein were first incubated for 8 to 10 mercaptoethanol. After 30 min at o”, lysis was completed by
min at 17”, and RNA polymerase was then added and the mixture raising the temperature gradually to 32” over a period of 10 to
incubated for another 20 min at 17”; RNA synthesis was initiated by 15 min. Magnesium acetate was added to 10 mM, and pan-
the addition of the four nucleoside triphosphates together with rif-
creatic DNase to 2 pg/ml. After the viscosity was substantially
ampicin and the mixture was incubated at 30”. For measurements of
total RNA synthesis, the reaction was terminated after 10 min by the reduced (5 to 10 min), 50 ml of 4 M NaCl were added, and the
addition of trichloroacetic acid to 5%, and the amount of RNA syn- lysate was centrifuged for 4 h at 30,000 rpm in a Spinco 30
thesis was determined by acid-insoluble radioactivity retained on a rotor. The supernatant fraction (550 ml) was dialyzed for 3 h
Millipore filter after filtration and washing with 5% trichloroacetic (two changes) against 5 liters of 10 mM KPO, (pH 6.41, 0.2 mM
acid and 70% ethanol.
For analysis of RNAs made, RNA synthesis was carried out in a EDTA, 0.2 mM dithiothreitol, 5% glycerol (Buffer A) contain-
0.2-ml reaction mixture in the presence of p factor (4 yglml) and [(u- ing 0.1 M KC1 (Fraction I).
32PlUTP. After lo-min incubation at 30”, 5 pg of pancreatic DNase Phosphocellulose Chromatography - Fraction I was applied
(“RNase-free”) and 150 pg of E. coli tRNA were added and the at a flow rate of 3 ml/min to a phosphocellulose column (go-ml
mixture was kept for 20 min at 0”. RNA was purified by phenol
extraction and three precipitation steps with 2 volumes of ethanol in
bed volume) equilibrated with Buffer A containing 0.1 M KCI.
the presence of 200 mM potassium acetate (pH 5.5). The precipitated
The column was washed with 150 ml of Buffer A containing 0.1
RNA was resuspended in 0.05 ml of 80 mM Tris/HCl (pH 8.3), 2.5 mM M KCl, and then eluted at a flow rate of 0.8 ml/min with a
EDTA, 8 M urea, and subjected to electrophoresis in a 3.5% poly- linear gradient (500 ml total volume) from 0.1 to 1.0 M KC1 in
acrylamide slab gel at 50 V for 10 h in the same buffer. The RNA Buffer A. Cro protein, showing the DNA-binding activity
bands were identified by autoradiography using x-ray film (Kodak
RP-54) as described by Rosenberg et al. (18). specific for A-DNA, eluted at a KC1 concentration of approxi-
Miscellaneous Methods - Polyacrylamide gel electrophoresis of de- mately 0.45 M (Fraction II). Binding specificity for A-DNA was
natured protein was carried out in 10% polyacrylamide and 0.1% checked by parallel assays with both A-DNA and himm434-
 Cro Protein of Phage h 6179
I
DNA, which lacks the specific binding sites for Cro protein
soo (0) o.6 ,,*' 0.16
(12). t r - _' ,' i
Sephadex G-75 Gel Filtration- Fraction II was concen-
trated by (NH&SO, precipitation (70% saturation at pH 6.0 600 c
for 20 min), centrifugation for 20 min at 13,000 r-pm, and

0?400
resuspension in 3 ml of 10 mM Tris/HCl (pH 7.31, 0.2 mM
EDTA, 0.2 rnM dithiothreitol, 5% glycerol (Buffer B) contain-
ing 0.2 M NaCl. The concentrated protein solution was applied -i‘, 200
to a column of Sephadex G-75 (90 x 2.5 cm) equilibrated
Buffer B containing 0.2 M NaCl, and eluted with the same
with
.Z:
buffer at a flow rate of 15 ml/h. The DNA-binding
specific for X-DNA eluted between 260 and 300 ml (approxi-
activity
?; 010
,= I
mately the position where a marker myoglobin protein elutedl ; z1
0 800 0.,6 ,”
(Fraction III). .-F E
DNA-cellulose Chromatography- Fraction III was diluted P 9
to 0.1 M NaCl with an equal volume of Buffer B and applied to 0 600 0.12 2
a h-DNA-cellulose column (lo-ml bed volume; 5 mg of A-DNA) 2
equilibrated with Buffer B containing 0.1 M NaCl. The column 400 0.08
was washed with 20 ml of Buffer B containing 0.1 M NaCl and
eluted with a linear gradient (50 ml total volume! from 0.1 M to
zoo
1.0 M NaCl in Buffer B. The DNA-binding activity specific for 0.04

A-DNA eluted at a NaCl concentration of approximately 0.3 M

(Fig. 2a) (Fraction IV). Fraction IV was diluted to 0.1 M NaCl 0 0

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with Buffer B and rechromatographed on h-DNA-cellulose (5- Fraction number
ml bed volume) with a linear gradient (30 ml total volume) FIG. 2. DNA-cellulose chromatography of Cro protein. Chroma-
from 0.1 to 1.0 M NaCl in Buffer B (Fig. 2b). The pooled tography of Fraction III from Sephadex gel filtration gave the elution
fractions, 18 to 21 (Fraction V), were used for the further profile shown in a. The fractions containing A-specific DNA-binding
studies described in this paper. Fraction V was free of DNase activity (indicated by the horizontal line) were pooled, diluted to 0.1
M NaCl, and rechromatographed on DNA-cellulose, giving the elu-
activity, as judged by sedimentation of 0.6 pg of A-DNA in an tion profile shown b. O-0, DNA-binding activity for A-DNA;
alkaline sucrose gradient after prior incubation with 0.6 pg of O-O, DNA-binding activity for Aimm434-DNA; A-A, A,,,.
Cro protein for 30 min at 30”. Fraction V was also free of RNase
activity, as judged by no detectable release of acid-soluble
radioactivity when 0.01 pg of A-13H]RNA was incubated for 15
min at 30” with 0.5 pg of Cro protein or by no change in the
1
size of 4 S and 6 S RNA analyzed by polyacrylamide gel
electrophoresis. Cro protein was stored at -20” in Buffer B
with 0.4 M NaCl and 50% glycerol without loss of activity over
a l-year period.
Properties of Altered Cro Protein Produced by cro- Muta-
tion - Although the binding specificity and the conditions for
synthesis provided a strong indication that the DNA-binding
protein purified above is the product of the cro gene, we have
also characterized an altered protein produced by a phage with
a missense mutation in the cro gene in order to complete the
identification of the Cro protein. For this purpose 2-liter cul-
tures of E. coli CGOOSu- were infected with cro+ or cro- (tof2
mutation)’ phage at 30” for 60 min, and a smaller scale puriti-
cation was carried out by phosphocellulose chromatography, Temperature, OC

followed by concentration with dry Sephadex G-75 and sedi- FIG. 3. Temperature sensitivity of Cro protein specified by mu-
mentation in a 10 to 30% glycerol gradient in Buffer A contain- tant cro gene. DNA-binding assays were carried out in duplicate as
described under “Methods,” except that the binding mixture was
ing 0.4 M KCl. Even when assayed at O”, the specific activity of incubated for 10 min at the temperature indicated on the figure
the mutant Cro protein thus isolated was about 15 to 20% that before filtration. The nonspecific binding to himm434-DNA has been
of wild type Cro protein, as judged by the amount of Cro subtracted from the binding to A-DNA to give the data presented in
protein determined by polyacrylamide gel electrophoresis in the figure. The binding to Aimm434-DNA at 0” was 3% with the Cro’
the presence of sodium dodecyl sulfate (both preparations preparation and 11% with the Cro- preparation; this varied only
slightly (by ~4%) with temperature. Thus the “background” correc-
contained about 60 to 70% Cro protein). tion is not responsible for the rapid change in the slope of the Cro-
The normal and mutant Cro proteins were characterized for binding curve. O-O, DNA binding for normal (cro+) protein;
sensitivity of the DNA-binding reaction to elevated tempera- O-O, DNA binding for cro” mutant protein.
ture (Fig. 31 and ionic strength (Fig. 4). There is a more severe
inhibitory effect of both temperature and ionic strength on the DNA-binding activity of the protein produced by the cro-
phage. From these results and others (see Ref. 12 and below),
’ tof2 is a temperature-sensitive cro mutation (15) and shows
temperature-dependent overproduction ofh-exonuclease (Y. Takeda, we conclude that the DNA-binding activity specific for A-DNA
unpublished results). that we are studying is the product of the A cro gene.
6180 Cro Protein of Phage h

--
(al 0.05M KCI

60 -

40 -

$ 20-
:
0
0; 0

z (b) 0.5M KCI

mu

FIG. 4. Salt sensitivitv of Cro urotein suecified bv mutant cro

gene. DNA binding assays were-carried out as described under
“Methods,” except that the binding mixtures included the KC1 con-
centration indicated on the figure. The nonspecific binding to
OC
0 5 10
Fraction
15
number
A 20 25

himm434-DNA has been subtracted from the binding to A-DNA to

give the data presented in the figure. The binding to himm434-DNA FIG. 5 (left). Velocity sedimentation of native Cro protein in a

Downloaded from www.jbc.org by on December 13, 2008

at 0.03 M KC1 was 3% with the Cro+ preparation and 11% with the glycerol gradient. Cro protein (0.2 ml of Fraction V) was layered on a
Cro- ureuaration: this decreased linearlv with increased salt concen- 10 to 30% glycerol gradient in Buffer B containing either 0.05 M KC1
tration and was 6% for Cro+ and 7% for”Cro-, respectively, at 0.23 M (a) or 0.5 M KC1 (5) and sedimented for 24 h at 49,000 r-pm. The
KCl. O-O, DNA binding for normal (cro+) protein; O-O, DNA vertical arrows denote the sedimentation position of marker proteins:
binding for cro- mutant protein. B is bovine serum albumin (M, = 65,000); 0, ovalbumin (45,000); C,
chymotrypsinogen (25,000); M, myoglobin (17,000). O-O, DNA-
binding activity for X-DNA; +O, DNA-binding activity for
Physical and Chemical Properties of Cro Protein Aimm434-DNA.
FIG. 6 (right). Electrophoresis of denatured Cro protein in a poly-
Physical Structure-To estimate the molecular weight of acrylamide gel containing sodium dodecyl sulfate. Cro protein (7 pg
native Cro protein, we carried out velocity sedimentation in a of Fraction V) was treated with 1% sodium dodecyl sulfate and 2% 2-
10 to 30% glycerol gradient (Fig. 5). Two salt concentrations mercaptoethanol for 16 h at room temperature, and electrophoresis
in 10% nolvacrvlamide and 0.1% sodium dodecvl sulfate was carried
were used in an effort to determine the stability of the subunit out for !P/i h ai 8 mA/tube as described by Weber and Osborn (19).
structure. In both 0.05 M KC1 and 0.5 M KCl, Cro protein has Protein was stained with Coomassie brilliant blue. The molecular
an estimated sedimentation coefficient of 1.9 to 2.0 S, as judged weight was estimated from marker proteins (bovine serum albumin,
by the sedimentation of marker proteins of known molecular ovalbumin, chymotrypsinogen, myoglobin, and cytochrome c) run in
weight. This indicates a molecular weight of 15,000 to 20,000, a parallel gel.
assuming that the axial ratio is in a typical range for a
globular protein (22). We wanted to determine the dissociation constant for Cro and
To determine the monomer molecular weight and estimate compare it to the very low value of lo-l3 M estimated for c1
the purity of the final preparation, we used polyacrylamide gel protein.
electrophoresis in sodium dodecyl suiiate. Only a single pro- In order to analyze the binding data, we needed to know
tein species was found when 7 pg of the preparation of Cro that Cro binding is sufficiently specific for the operator sites on
protein were used (Fig. 6); after electrophoresis of 28 pg, three A-DNA for this interaction to dominate the binding curve and
faint minor bands were discernible. From these results we that one active Cro protein is sufficient to retain the radioac-
judge the preparation to be more than 95% Cro protein. The tive A-DNA on the nitrocellulose filter in the standard binding
monomer molecular weight of Cro protein is estimated to be assay. This information is provided by the binding curve of
approximately 9000 from the migration of marker proteins of Fig. 7, in which Cro concentration is varied for two DNA
known molecular weight. substrates, A and Aimm434; Aimm434 is mainly identical with
Amino Acid Composition-The amino acid composition of A but lacks the region of A-DNA containing the specific bind-
Cro protein is presented in Table II. The composition is high in ing sites for c1 and Cro proteins (7, 10, 12). The binding to A-
lysine and alanine and lacks cysteic acid and tryptophan, DNA is linear at low concentrations of Cro protein, indicating
showing an interesting similarity to the prokaryotic DNA- that 1 active Cro molecule can retain 1 A-DNA mo1ecule.2 The
binding protein HU (23,241 and to the eukaryotic histone H2B binding is also largely specific for A-DNA. The “nonspecific”
(25). From the amino acid composition, the monomer molecu- binding that we have observed for Aimm434-DNA has also
lar weight is determined to be 8600. From the combined physi- been found for other DNA-binding regulatory proteins (26,271,
cal and chemical studies, we conclude that native Cro protein and presumably represents relatively weak binding interac-
is probably a dimer of identical subunits. tions that can occur anywhere on a DNA molecule (a similar
binding curve to that of Aimm 434 has been found also for (P80
Binding Properties of Cro Protein DNA). From the data of Fig. 7, we conclude that the standard
Equilibrium Binding- Previous experiments have shown * Because of the relatively large dissociation constant of Cro pro-
that Cro protein binds to the same operator region used by the tein, the washing procedure used for the filter assay is important
A c1 protein, the “A repressor” that maintains lysogeny (12). (see “Methods”).
 Cro Protein of Phage h 6181
TABLE II
Amino acid composition of Cro protein
mollunit” mol %
Alanine 9 11.5
Arginine 4 5.1
Aspartic acid + asparagine 7 10.0
Cysteic acid 0 0.0
Glutamic acid + glutamine 6 7.7
Glycine 5 6.4
Histidine 1 1.3
Isoleucine 6 7.1
Leucine 4 5.1
Lysine 10 12.8
Methionine 2 2.6
Phenylalanine 3 3.8
Proline 3 3.8
Serine 4 5.1
Threonine Of I 1 1 I
7 10.0 0 5.0 7.5 10.0
2.5
Tryptophan 0 0.0 DNA concentration, pg/ml
Tyrosine 3 3.8
Valine 4 5.1 FIG. 8. DNA binding of Cro protein as a function of DNA concen-
tration. Binding assays were carried out as for Fig. 7, except that h-
DNA concentration was varied as indicated on the figure and Cro
Total 78
concentration was held constant at 15 rig/ml (O---O) or 10 rig/ml
o Calculations were made using the assumption that the protein (0-O).

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has 1 histidine residue per monomer unit.

- - I I t I I I I
0 20 40 60 0 2 4 6
Cro concentration, rig/ml Time, min

FIG. 7. DNA binding of Cro protein as a function of Cro concen- FIG. 9. Stability of Cro.h-DNA complex. A-13*P1DNA (0.5 pg/ml)
tration. Binding assays were carried out using the standard binding was incubated with Cro protein for 10 min at 0” at a binding level of
conditions (DNA concentration, 2 pglml), but the filter was washed 20% of the input DNA (zero time), unlabeled A-DNA was added to 15
with 0.5 ml of 45% ethanol as described under “Methods.” O-O, pglml, and O.l-ml aliquots were taken out at intervals and filtered to
DNA-binding activity for h-DNA; O-O, DNA-binding activity for determine the remaining A-[32PlDNA.Cro complex. O-O, at pH
himm434-DNA. 7.3; O---O, at pH 6.6.

DNA-binding assay will allow us to estimate specific binding to interpret because the measurements are subject to substan-
constants. tial variation with ionic strength, temperature, and pH. How-
A binding curve in which DNA concentration is varied is ever, Cro does appear to be a substantially weaker DNA-
most appropriate for the measurement of an equilibrium disso- binding protein than other specific regulatory proteins studied
ciation constant (28, 29); the results of this experiment for Cro so far; approximate values for A c1 protein, lac repressor, and
protein are shown in Fig. 8. The detailed interpretation of the araC protein are lo-l3 M (7), lo-l3 M (281, and lo-‘* M (29),
binding curve is complicated by the fact that we do not know respectively.Y
precisely how many specific binding sites for Cro are present Dissociation Rate Constant - To estimate the dissociation
on a A-DNA molecule. For A c1 protein, there are three binding rate constant, we formed a Cro.X-13’PlDNA complex and mea-
f”operator”) sites on either side of the cI gene, termed ~a,, oa2, sured the loss of X-1321DNA from this complex with time in the
and or,:) and oi,,, oL2, and oL3; of these oR, and oL, have substan- presence of a 30-fold excess of unlabeled A-DNA (Fig. 9). Since
tially higher affinities for c1 than the others (30). For Cro a Cro molecule should not reassociate to a significant extent
protein, we only know so far that Cro binds to at least two sites with the 132P1DNA once released, the initial decrease in
in the 0,. and oH region (see Ref. 12 and below). If we assume [32P]DNA bound should follow an exponential first order decay
two binding sites per 3 x lo7 daltons of A-DNA (311, we :I Since the number of binding sites for Cro is at least two, an
calculate a dissociation constant of 8 to 9 x 10-l’ M. Compari- underestimate of the number of Cro sites will only increase the
sons with dissociation constants of other proteins are difficult differences between Cro and other specific regulatory proteins.
 Cro Protein of Phage A

8-9S(
a
$ 25

0 4 8 12 6S--,
Cro protein, pg/ml
FIG. 10. Repression of total RNA synthesis by Cro protein. RNA
synthesis was carried out using purified RNA polymerase and hb2 or
hb2imm434-DNA as a template as described under “Methods.” The
DNA was first incubated with the levels of Cro protein indicated on
the figure, RNA polymerase was then added, and RNA synthesis 4s-+ :
was initiated by the addition of four nucleoside triphosphates to- a b c
gether with rifampicin. Without Cro protein, 16 and 9 pmol of
FIG. 11. Effect of Cro protein on individual RNA chains. RNA
13HlUMP were incorporated for A- and Aimm434-DNA, respectively,

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synthesis was carried out in the presence of p factor using 13*PlUTP.
during the incubation for 10 min at 30”. O--O, RNA synthesis with
A-DNA; O-O, RNA synthesis with Aimm434-DNA. The 13*PlRNA was extracted, subjected to electrophoresis in a 3.5%
polyacrylamide slab gel containing 8 M urea, and identified by
autoradiography as described under “Methods.” a, A-DNA, no Cro
curve. Because of the rapid decay found under our standard protein; b, A-DNA, Cro protein added at 3 pg/ml; c, Aimm434-DNA,
Cro protein added at 3 pg/ml.
binding condition of pH 7.3, we also carried out binding assays
at pH 6.6. The calculated dissociation rate constant is about 2 TABLE III
x lo-* s-l at pH 7.3 and 5 x 10m3 s-l at pH 6.6. As expected Comvetition between Cro and RNA _polvmerase
-
from the equilibrium binding data, the half-life for Cro disso- Proteins and order of addition
L3HlUMP in- Repres-
ciation at pH 7.3 is much less than that found for c1 protein corporated sion
and lac repressor, and substantially less than the 3-min value pITlO %
found for araC protein. In an experiment carried out under RNA polymerase” 16.5 0
identical conditions, we found the half-life for c1 dissociation to Cro and then RNA polymeraseb 3.2 81
RNA polymerase and Cro togetherc 3.5 79
be greater than 100 min. We have also attempted to measure
RNA polymerase and then Crod 16.4 1
the dissociation rate for “nonspecific” binding, using DNA
lacking the specific operator sites (kimm434- or @80-DNA). a RNA polymerase was incubated with A-DNA at 17” for 20 min
The dissociation was very rapid with a half-life of 510 s, which before addition of nucleoside triphosphates and rifampicin (see
is consistent with the concept that the nonspecific binding is a “Methods”).
substantially weaker interaction than the specific binding to * Cro protein was added to A-DNA for 10 min at 17” followed by an
RNA polymerase incubation as for Line 1.
the regulatory sites.
c Cro protein and RNA polymerase were added to A-DNA together
We have attempted to determine the association rate con- followed by an incubation at 17” for 30 min.
stant, but have not been able to do so because Cro protein is d RNA polymerase was incubated with A-DNA as for Line 1, and
unstable at the very high dilution required for this measure- Cro protein was then added for 10 min at 17” before addition of
ment. If the rate constant for Cro is comparable to that esti- nucleoside triphosphates.
mated for araC protein (2 x 10y M-’ SK’), the value calculated
for the equilibrium dissociation constant is lo-” M at pH 7.3. radiography (Fig. 111. The RNA produced in vitro from A-DNA
in the presence of p factor is predominantly of four size classes,
Effect of Cro Protein on RNA Synthesis designated 4 S, 6 S, 8 to 9 S, and 12 S (14,18,32,33). The 8 to 9
From experiments carried out in vivo, we expect Cro protein S and 12 S transcripts represent RNA chains initiated at thep,
to act as a specific repressor of RNA synthesis initiated at the andp, promoters, respectively, and the 4 S and 6 S transcripts
early promoter sites of A-DNA, pL and pR (see Refs. 1 to 6 and represent promoters near the ~11 and Q genes, respectively
Fig. 1). To test this expectation, we used purified A-DNA as a (Fig. 1). The results of Fig. 11 show that Cro protein represses
template for RNA polymerase and studied the effect of Cro 8 to 9 S and 12 S RNA synthesis from A-DNA but not from
protein on RNA synthesis. To ensure that Cro effects derive Aimm434-DNA; 6 S RNA synthesis was not repressed by Cro
from the specific binding reaction, we used Ximm434-DNA as a with A-DNA as a template (although not shown at this gel
template in parallel experiments. exposure, 4 S RNA was also not repressed by Crol. As expected
The capability of Cro protein to function as a specific repres- from the failure of Cro to repress total RNA synthesis from
sor in vitro is shown in Fig. 10, in which strong repression of Aimm434-DNA (Fig. lo), there was no difference in the gel
total RNA synthesis from A-DNA occurs in the absence of any pattern of RNA synthesis from Aimm434-DNA in the presence
repression effect on RNA synthesis from Aimm434-DNA. The or absence of Cro (data not shown).
RNA products were analyzed further by separation through If the repression of transcription noted in Fig. 10 and Fig. 11
polyacrylamide gel electrophoresis and visualization by auto- occurs at the initiation step of RNA synthesis, the binding of
 Cro Protein of Phage A 6183

RNA polymerase to DNA before Cro might be expected to these proteins will be an interesting study in protein evolu-
abolish repression. Table III shows this is the case (compare tion.
Lines 2 and 4). Cro added at the same time as RNA polymerase
is an effective repressor (Table III, Line 3), presumably be- Acknowledgments-We thank Drs. P. Miller and D. Cole
cause of the relatively slow formation of a tight binding initia- for performing the amino acid analysis of Cro protein, and
tion complex by RNA polymerase (see Ref. 341. Thus, Cro Drs. J. Galluppi and J. Richardson for the gift of termination
probably represses RNA s.ynthesis by blocking the capacity of factor p
RNA polymerase to bind at the promoter site; a similar mecha- REFERENCES
nism has been inferred for c1 protein (7-11). 1. Echols, H. (1972) Annu. Reu. Genet. 6, 157-190
From these results, we conclude that Cro is an effective and 2. Herskowitz, I. (1973) Annu. Rev. Genet. 7, 289-324
3. Echols. H. (1974) Biochimie 56. 1491-1496
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sis from the early promoters, pL and pR. Since 8 to 9 S RNA is 5. Takeda, Y., Matsubara, K., and Ogata, K. (1975) Virology 65,
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7. Chadwick, P., Pirrotta, V., Steinberg, R. A., Hopkins, N., and
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283-294
Cro protein has a special role among the specific regulatory 8. Steinberg, R. A., and Ptashne, M. (1971) Nature New Biol. 230,
proteins analyzed so far because it functions during a temporal 276-280
%vitch” in viral development from a replication-oriented Wu, A. M., Ghosh, S., Willard,
9. M., Davison, J., and Echols, H.
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because the cro gene is transcribed during the earliest (“imme- H. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2249-2253
diate early”) stage of RNA synthesis (l-5). Our biochemical 13 Burgess, R. R., and Jendrisak, J. J. (1975) Biochemistry 14,
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14 Roberts, J. W. (1969) Nature 224, 1168-1174
result from the relatively low affinity of Cro protein for its 15 Takeda, Y., Oaata. K., and Matsubara. K. (1975) Virolopv I” 65.
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repressor only after the time required for the synthesis of a 16 Alberts, B., and Herrick, G. (1971) Methods Enzymol. 21, 198-
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17 Botchan, P. (1976) J. Mol. Biol. 105, 161-176
In contrast to Cro, the c1 protein of phage A functions as a 18 Rosenberg, M., decrombrugghe, B., and Weissman, S. (1975) J.
steady state repressor to maintain lysogeny through a com- Biol. Chem. 250, 4755-4764
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the different biological requirements for the repression of 20. Beaven, G: H., and Holiday, E. R. (1952) in Aduance~ in Protein
early genes during lytic growth or lysogeny has led to the Chemistry (Anson, M. L., Bailey, K., and Edsall, J. T., eds)
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evolution of biochemically different repressors, a high affinity 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.
maintenance repressor (~1) and a low affinity lytic repressor (1951) J. Biol. Chem. 193. 265-275
(Cro), although both use the same operator region of h DNA. 22. Smith, M. H. (1968) in Handbook OfBiochemistry (Sober, H. A.,
Cro and c1 also appear to differ in their effect on transcription ed) pp. C-10-23, The Chemical Rubber Co.. Cleveland. Ohio
23. Rouvikre-Yaniv, J., and Gros, F. (1975)Proc.‘Natl. Acad; Sci. U.
of the cro and c1 genes. Because they repress transcription S. A. 72, 3428-3432
initiated at pro both c1 and Cro are repressors of the CM gene 24. Haselkorn, R., and Rouviere-Yaniv, J. (1976) Proc N&l. Acad.
(thus Cro is an “autorepressor”); however, c1 can function Sci. U. S. A. 73, 1917-1920
either as a repressor or an activator of the c1 gene transcript 25. Iwai, K., Ishikawa, K., and Hayashi, H. (1970) Nature 226,1056-
initiated at pM, whereas Cro probably functions only as a 1058
26. Lin. S.-Y.. and Rices. A. D. (1975) Cell 4, 107-112
repressor of this RNA (Fig. 1) (l-12, 30). This additional 27. Van Hippcl, P. H.ykkvzin, A., Gross, C. A., and Wang, A. C.
functional difference between the two repressors should be (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4808-4812
clarified by a more detailed analysis of binding and transcrip- 28. Riggs, A. D., Suzuki, H., and Bourgeois, S. (1970) J. Mol. Biol.
tion. 48, 67-83
29. Wilcox. G.. Clemetson. K. J.. Clearv, P.. and Enalesberz, E.
Cro protein is the smallest DNA-binding regulatory protein (197k)J.‘Mol. Biol. 85, 589-602 ” - -
purified so far that exhibits specificity for a DNA site. Re- 30. Ptashne, M., Backman, K., Humayun, M. Z., Jeffrey, A.,
markably the amino acid composition of Cro protein is very Maurer, R., Meyer, B., and Sauer, R. T. (1976) Science 194,
similar to that of the transcription factor TFl of phage SPOl 156-161
(351, the prokaryotic DNA-binding protein HU (23, 241, and 31. Davidson, N., and Szybalski, W. (1971) in The Bacteriophage
Lambda (Hershey, A. D., ed) pp. 45-82, Cold Spring Harbor
the eukaryotic histone H2B (25); the similarity to HU protein Laboratory, Cold-Spring Harbor, New York
is mosl striking. HU is also a small protein (molecular weight 32. Blattner, F., and Dahlberg, J. (1972) Nature New Biol. 236, 227-
7000) but lacks any known specificity for DNA sites (231. An 232
interesting possibility is that HU protein might have a mini- 33. Roberts, J. W. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 3300-
3304
mal structure for recognition of double-stranded DNA, to Chamberlin, M. J. (1974) Annu. Reu. Biochem. 43, 721-775
34.
which Cro protein has added a minimal structure for specific 35. Johnson, G. G., and Geiduschek, E. P. (1972)5. Biol. Chem. 247,
sequence recognition. A comparable structural analysis of 3571-3578