Screening and detection of scFV fragments specific to HA and NP protein of H5N1 virus
Cro Regulatory Protein h5n1
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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 HARRISON ECHOLS
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 lyti c growth. We have
purified Cro protein to virtual homogeneity and analyzed itsstructure and properties as a DNA-binding protein and re-
pressor of RNA synthes is. To confirm that the protein is theproduct of the cro gene, we have also shown that a missense
mutation in the ero gene leads to a product that is more
temperature- and salt-sensitive in its DNA-binding property.
As purified, Cro protein is a dimer o f identical subunits ofmolecular weight 8600. The purified protein binds to A-DNA
carrying the specif ic binding sites (operators oL and oli) withan estimated dissociation constant of lo-‘” M to lo-” M; there
is also weaker binding to other si tes on DNA, as found forother DNA-binding regulatory proteins. In a purified tran-
scription system, the Cro protein is an effect ive and speci fic
repressor of RNA synthesis from the N and cro genes; thus
Cro is an autorepressor which regulates its own synthesis. Acomparison of the properties of the two A repressor proteins,
CI and Cro, indicates that cI is a “strong repressor” special-
ized for complete turnoff of lyti c functions needed for themaintenance of lysogeny, whereas Cro is a “weak repressor”
specialized for a gradual turnoff of early viral genes that
potentiates the late stage of lyt ic development.
The temperate bacteriophage A spec ifies two repressor pro-
teins, c1 and Cro, which carry out regulatory functions essen-
tial fo r different aspects o f the viral life cycle. The c1 protein
acts under conditions of stable lysogeny to maintain repression
of the integrated viral DNA; Cro protein acts during lyt ic
development to turn of f the expression of the phage genesactive early after infection (l-6) and thus potentiates the
early-late switch in expression of viral genes.
The c1 protein has been purified and extens ively character-
ized in vitro for binding to speci fic operator sites on X-DNA and
ability to repress RNA synthesis initiated at the X promoter
sites active early during viral development (7-11) (Fig. 1). In aprevious paper, we presented data indicating that Cro protein
is a DNA-binding protein which binds to the same operator
region of A-DNA as does c1 (12). This report describes the
purification of Cro protein to apparent homogeneity, presents
a more detailed characterization of its structure and DNA-
* This research was supported in part by Grant GM 17078 from theNational Institute of General Medical Sciences.
$ Postdoctoral Fellow of the National Cancer Institute.
binding act ivi ty, and establishes the capaci ty of Cro to func-tion as a speci fic 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.
EXPERIMENTAL PROCEDURES
Materials
Nucleic Acids - Bacteriophage DNA was prepared from purifiedphage by phenol extraction as described previously (10, 12).“Chicken blood” DNA and Escherichia coli tRNA were obtained fromCalbiochem.
Proteins-E. coli RNA polymerase was prepared as described byBurgess and Jendrisak (13). Termination factorp, purified accordingto Roberts (141, was the gif t of J. Galluppi and J. Richardson,University of Indiana. Pancreatic DNase was obtained from Worth-ington and ovalbumin, chymotrypsin, myoglobin, and bovine serum
album in from Schwarz/Mann.
Bacterial and Phage Strains - The bacterial host used to prepare
Cro protein was CGOOSu- . The infecting phage for large scale prepa-
rations of wild type Cro protein w as hNam53ulu3cIaml4Sam7; to
characterize the temperature-sen sitive mutant protein, paralle l in-fections by hNam53ulu3cro+ and hNam53Num7ulo3cro~ were used, inwhich the cro- mutation was tof2 (15). The A mutations and the
rationale for using them have been described in more detail previ-
ously (12). In brief, N- mutation eliminates production of most h
proteins besides Cro, ulu3 may increase Cro production, cI- mutationeliminates the DNA-binding activity of c1 protein, and S- mutation
prevents cell lysis.Other Mate&Es- Cellulose (CFll) and phosphoc ellulose (PII)
were obtained from Whatma n and Sephadex G-75 (140 to 120 CL
particle size) from Pharmacia. h-DNA-cellulose was prepared as
describ ed by Alberts and Herrick (16). Ultrapure ammon ium sulfatewas obtained from Schwarz/Mann, acrylamide and sodium dodecyl
sulfate from Bio-Rad, unlabeled nucleoside triphosphates fromSigma, [5-“HIUTP from Radiochem ical Center, Amersham, I IY-
82P]UTP from New England Nuclear, and rifampicin from Lepetit.
Methods
DNA-binding Assay for Cro Protein- The standard assay was
done essentially as described previously (10, 12). The assay measuresthe retention of A-I”‘P]DNA on a nitroc ellulo se filter by virtue of its
tight bind ing to Cro protein; for purifica tion an exces s (loo-fold) of
unlabeled “chicken blood” DNA was added to compete for the bind-
ing of proteins that associate with DNA but lack specificity for A-
DNA. To further differentlate the operato r-specific Cro protein fromother h-DNA-binding proteins, we did parallel assays with h-DNA
and Aimm434-DNA (which is the same as A-DNA except for theoperator-containin g immun ity region). The standard assay mixture
contained 0.2 pg of A-I,L’PIDNA and 20 pg of chicken blood DN A in
0.1 ml of “binding buffer”: 10 rnM Tris/H Cl (pH 7.31, 20 mM KCl, 10
rnM MgCl,, 0.2 rnM dithioth reitol, and 0.2 rnM EDT A. After in cuba-
tion for 10 min at O”, the mixture was filtered with a nitrocellulose
6177
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6178 Cro Protein of Phage X
12s
Recomb.aenes CHIT22
‘IS
cm cII-Redn.aenes Q22
1M i-4
PL PR
OL
8
)
OR
CrO PM
FIG. 1. The activity of c1 and Cro inferred from expe riments in
uiuo and in vitro. To maintain the repression essential for lysogeny,the cI protein acts at operators oL and oH to repress leftward and
rightward transcriptio n initiate d at the early promoter site s pL andpR, respectively; the c1 protein also activates leftward transcriptio n
of the c1 gene initiated at the maintenance promoter phi The repres-sion activity of c1 keeps the entire phage genome repressed because
theN gene product is required as a positive regulator of other ge nes.
The Cro protein acts to turn off (or turn down) the expression of early
genes during the late stage of lytic development through its capacity
to act at oL and os to repress early gene trans cription; the Cro proteinprobably represses transcription of the c1 gene initiated at phi Other
genes and genetic regions on the figure are: ~11 and cIII, genes which
activate initial expression of the c1 gene during infection; Q, gene for
protein that activates late gene transcription; Repin, replication;
Recomb, recombination. The waved lines above the A genome indi-cate early RNAs observed in uiuo and in vitro; the S values indicate
approximate sedimentation coefficient of the RNAs.
filter (B6, Schleicher and Schuell), washed with 0.5 ml of bindingbuffer, dried, and the retained radioactivity was determined by
liquid scintillation counting. For assays involving purified Cro pro-
tein, the chicken blood DNA was omitted. One unit of DNA-bindingactivity is defined as the quantity sufficient to retain 1 pg of A-DNA
on the filter. The bindi ng values are corrected for a “background” of
DNA that is retained on the filter in the absence of binding proteins;
this varies from 5 to 15% with DNA preparation. For the experi-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-
ml wash with 45% ethanol. This procedure gave higher and more
reproducib le bindin g data with lower levels of Cro protein, presum-
ably because dissociation during the washing step did not occur (nodecrease 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
reducing the volume of the standard wash ing buffer (0.1 ml), but thedata were erratic with a higher level of background bindin g.
RNA Synthesis and Analysis-RNA synthesis was carried outusing purified RNA polymerase and hb2- or Ab2imm434-DNA in a
reaction in which RNA chains were initiated in the presence of
rifampicin; the use of DNA carrying the b2 deletion and of rifampicin
results in a larger fraction of RNA from the promoter site s on A-DNAused in viva for early RNA (10, 14, 17). The standard reaction
mixture (0.1 ml) contain ed: 100 rnM Tris/H Cl (pH 7.2), 40 mM NaCl,
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
A-DNA. The A-DNA and Cro protein were first incuba ted for 8 to 10
min at 17”, and RNA polymerase was then added and the mixture
incubated for another 20 min at 17”; RNA synthesis was initiated bythe addition of the four nucleoside triphosphates together with rif-
ampicin and the mixture was incubated at 30”. For measurements of
total RNA synthesis, the reaction was terminated after 10 min by the
addition of trichloroacetic acid to 5%, and the amount of RNA syn-
thesis was determined by acid-insoluble radioactivity retained on a
Millipore filter after filtration and washing with 5% trichloroaceticacid and 70% ethanol.
For analysis of RNAs made, RNA synthesis was carried out in a
0.2-ml reaction mixture in the presence of p factor (4 yglml) and [(u-
32PlUTP . After lo-min incubation at 30”, 5 pg of pancreatic DNase
(“RNase-free”) and 150 pg of E. co li tRNA were added a nd the
mixture was kept for 20 min a t 0”. RNA was purified by pheno lextraction and three precipitation steps with 2 volumes of ethanol in
the presence of 200 mM potassium acetate (pH 5.5). The precipitatedRNA was resuspended in 0.05 ml of 80 mM Tris/H Cl (pH 8.3), 2.5 mM
EDTA, 8 M urea, and subjected to electrophoresis in a 3.5% poly-acrylamide slab gel at 50 V for 10 h in the same buffer. The RNA
bands were iden tified by autoradiography usin g x-ray film (Kodak
RP-54) as described by Rosenberg et al. (18).
Miscellaneous Methods - Polyacrylamide gel electrophoresis of de-
natured protein was carried out in 10% polyacrylam ide and 0.1%
TABLE I
Purific ation of Cro protein
Fraction Volume Total protein Spz$tcyac- Yield
ml wunitsing
protein%
I. Crude extract 550 4455 -a -0
II. Phospho cellulose 50 39.5 157 100
III. Sephadex G-75 40 5.2 415 35
IV. DNA-cellulose 1 6 0.88 3355 48V. DNA-cellulose 2 4 0.60 4000 39
0 The binding is not specific for A-DNA in the crude extract, and so
no value for activity can be given at this stage.
sodium dodecyl su lfate as described by Weber and Osborn (19).
Velocity sedimentation was performed in 10 to 30% glycerol gra-
dients containing 10 mM Tris/HCl (pH 7.3), 0.2 mM EDTA , 0.2 mMdithiothreitol, and KC1 as noted; sedimentation was at 49,000 rpm
for 24 h in a Spinco SW 50.1 rotor. Amino acid analysis of Cro protein
was carried out with a Beckman analyzer after a 20-h hydrolysis of
the protein in 6 N HCl at 110” in an evacuated tube; analysis for
tryptophan was done spectroph otometrica lly by the method ofBeaven and Holiday (20). Protein was determined by the method of
Lowry et al. (21) with bo vine serum album in as a standard. Salt
concentrations in gradient fractions were measured with a conduc-
tivity meter (Radiometer type CDM2e).
RESULTS
Purific ation of Cro Protein
A summary of the purification of Cro protein is presented in
Table I. Unless otherwise noted, all operations were performed
at O-4”.
Growth of Znfected Cells-A culture (100 liters) of Esche-
richia coli CGOOSu- was grown in a New Brunswick Fermen-
ter at 30” in a broth conta ining 2% Difco Bacto-tryptcme, 1%
yeast extract, 0.5% NaCl, and 0.2% maltos e. When the A,!,,, of
the culture reached 1.0, MgCl, was added to 10 mM and h
phage (strain Nam53cIam14ulv3Sum7) was added at a multi-
plicity of 10 phages/cell. After 60 min at 30”, the cells were
harvested by centrifugation in a Sharples continuous flow
centrifuge, resuspended in 50 ml of 10% sucrose, 50 mM Tris/HCI (pH 7.8, quick-frozen, and stored at -20”. The procedure
yielded 150 g of cells.
Preparation of Extract-The frozen cells were thawed and
mixed with 80 ml o f 2 mg/ml of lysozyme in 250 mM Tris/HCl
(pH 7.51, 1 mM EDTA, and 19 ml of 4 M NaCl, 10 mM 2-
mercaptoethanol. After 30 min at o”, lys is was completed by
raising the temperature gradually to 32” over a period of 10 to15 min. Magnesium acetate was added to 10 mM, and pan-
creatic DNase to 2 pg/ml. After the viscos ity was substantially
reduced (5 to 10 min), 50 ml o f 4 M NaCl were added, and the
lysate was centrifuged for 4 h at 30,000 rpm in a Spinco 30rotor. The supernatant fraction (550 ml) was dialyzed for 3 h
(two changes) against 5 liters of 10 mM KPO, (pH 6.41, 0.2 mMEDTA, 0.2 mM dithiothreitol, 5% glycerol (Buffer A) contain-ing 0.1 M KC1 (Fraction I).
Phosphocellulose Chromatography - Fraction I was applied
at a flow rate o f 3 ml/min to a phosphocellulose column (go-ml
bed volume) equilibrated with Buf fer A containing 0.1 M KCI.The column was washed with 150 ml of Buf fer A containing 0.1
M KCl, and then eluted at a flow rate o f 0.8 ml/min with a
linear gradient (500 ml total volume) from 0.1 to 1.0 M KC1 in
Buffer A. Cro protein, showing the DNA-binding act ivi ty
specif ic for A-DNA, eluted at a KC1 concentration of approxi-
mately 0.45 M (Fraction II). Binding spec ifici ty for A-DNA was
checked by parallel assays with both A-DNA and himm434-
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Cro Protein of Phage h 6179
DNA, which lacks the specif ic binding sites for Cro protein
(12).Sephadex G-75 Gel Filtration- Fraction II was concen-
trated by (NH&SO, precipitation (70% saturation at pH 6.0
for 20 min), centrifugation for 20 min at 13,000 r-pm, and
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
to a column of Sephadex G-75 (90 x 2.5 cm) equilibrated withBuffer B containing 0.2 M NaCl, and eluted with the same
buf fer at a flow rate of 15 ml/h. The DNA-binding act ivi ty
specif ic for X-DNA eluted between 260 and 300 ml (approxi-
mately the position where a marker myoglobin protein elutedl
(Fraction III).DNA-cellulose Chromatography- Fraction III was diluted
to 0.1 M NaCl with an equal volume of Buffer B and applied to
a h-DNA-cellulose column (lo-ml bed volume; 5 mg of A-DNA)
equilibrated with Buffer B containing 0.1 M NaCl. The columnwas 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 to1.0 M NaCl in Buffer B. The DNA-binding act ivi ty specif ic for
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
with Buffe r B and rechromatographed on h-DNA-cellulose (5-
ml bed volume) with a linear gradient (30 ml total volume)from 0.1 to 1.0 M NaCl in Buffer B (Fig. 2b). The pooled
fractions, 18 to 21 (Fraction V), were used for the further
studies described in this paper. Fraction V was free of DNase
act ivi ty, as judged by sedimentation of 0.6 pg of A-DNA in an
alkaline sucrose gradient after prior incubation with 0.6 pg of
Cro protein for 30 min at 30”. Fraction V was also free of RNaseact ivi ty, 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
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 act ivi ty over
a l-year period.Properties of Altered Cro Protein Produced by cro- Muta-
tion - Although the binding spec ific ity and the conditions forsynthesis 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,
followed by concentrat ion with dry Sephadex G-75 and sedi-
mentation in a 10 to 30% glycerol gradient in Buffer A contain-
ing 0.4 M KCl. Even when assayed at O”, the specific activ ity of
the mutant Cro protein thus isolated was about 15 to 20% thatof wild type Cro protein, as judged by the amount of Cro
protein determined by polyacrylamide gel electrophoresis in
the presence of sodium dodecyl sulfate (both preparations
contained about 60 to 70% Cro protein).
The normal and mutant Cro proteins were characterized for
sens itiv ity of the DNA-binding reaction to elevated tempera-
ture (Fig. 31 and ionic strength (Fig. 4). There is a more severe
inhibitory eff ect of both temperature and ionic strength on the
’ tof2 is a temperature-sensitive cro mutation (15) and shows
temperature-dependent overproduction ofh-exonu clease (Y. Takeda,unpublished results).
I
soot
(0) o.6 ,,*'
r,'
i0.16
- _'
600 c
?4000-i‘, 200.Z:; 0,= 10
; z10 800
F0.,6 ,”
E-
P 90 600 0.12 2
2
400 0.08
zoo 0.04
00
Fraction number
FIG. 2. DNA -cellulo se chromatography of Cro protein. Chroma-
tography of Fraction III from Sephadex gel filtration gave the elutio n
profile shown in a. The fractions containing A-specific DNA-bindingactivity (indica ted by the horizontal line) were pooled , diluted to 0.1
M NaCl, and rechromatographed on DNA-cellulose, giving the elu-tion profile shown b. O-0, DNA -binding activity for A-DNA;
O-O, DNA-b inding activity for Aimm434 -DNA; A-A, A,,,.
1
Temperature, OC
FIG. 3. Temperature sensitiv ity of Cro protein spe cified by mu-
tant cro gene. DNA-binding assays were carried out in duplicate as
describ ed u nder “Methods,” except that the bindi ng mixture was
incubated for 10 min at the temperature indicated on the figure
before filtration. The nonspe cific binding to himm434-DNA has beensubtracted from the bindin g to A-DNA to give the data presented in
the figure. The binding to Aimm434-DNA at 0” was 3% with the Cro’preparation and 11% with the Cro- preparation; this varied only
sligh tly (by ~4%) with temperature. Thu s the “background” correc-
tion is not responsible for the rapid change in the slope of the Cro-
bindi ng curve. O-O, DNA bindin g for normal (cro+) protein;
O-O, DNA bindi ng for cro” mutant protein .
DNA-binding act ivi ty of the protein produced b y the cro-
phage. From these results and others (see Ref . 12 and below),
we conclude that the DNA-binding act ivi ty speci fic for A-DNA
that we are studying is the product of the A cro gene.
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6180 Cro Protein of Phage h
FIG. 4. Salt sens itiv itv of Cro urotein suecified bv mutant crogene. 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 tohimm434-DNA has been subtracted from the binding to A-DNA to
give the data presented in the figure. The binding to himm434-DNAat 0.03 M KC1 was 3% with the Cro+ preparation and 11% with theCro- ureuaration: this decreased linearlv with increased salt concen-tration and was 6% for Cro+ and 7% for”Cro-, respectively, at 0.23 M
KCl. O-O, DNA binding for normal (cro+) protein; O-O, DNAbinding for cro- mutant protein.
Physical and Chemical Properties of Cro Protein
Physical Structure-To estimate the molecular weight of
native Cro protein, we carried out velocity sedimentation in a
10 to 30% glycerol gradient (Fig. 5). Two salt concentrations
were used in an effort to determine the stability of the subunit
structure. In both 0.05 M KC1 and 0.5 M KCl, Cro protein has
an estimated sedimentation coeff icient of 1.9 to 2.0 S, as judgedby the sedimentation of marker proteins of known molecular
weight. This indicates a molecular weight of 15,000 to 20,000,assuming that the axial ratio is in a typical range for a
globular protein (22).
To determine the monomer molecular weight and estimatethe purity of the final preparation, we used polyacrylamide gel
electrophoresis in sodium dodecyl suiiate. Only a single pro-tein species was found when 7 pg of the preparation of Cro
protein were used (Fig. 6); after electrophoresis of 28 pg, three
faint minor bands were discernible. From these results we
judge the preparation to be more than 95% Cro protein. The
monomer molecular weight of Cro protein is estimated to be
approximately 9000 from the migration of marker proteins of
known molecular weight.
Amino Acid Composition-The amino acid composition o f
Cro protein is presented in Table I I. The composition is high inlysine and alanine and lacks cysteic acid and tryptophan,showing an interesting similarity to the prokaryotic DNA-
binding protein HU (23,241 and to the eukaryotic histone H2B
(25). From the amino acid composition, the monomer molecu-
lar weight is determined to be 8600. From the combined physi-
cal and chemical studies, we conclude that native Cro protein
is probably a dimer of identical subunits.
Binding Properties of Cro Protein
Equilibrium Binding- Previous experiments have shown
that Cro protein binds to the same operator region used by the
A c1 protein, the “A repressor” that maintains lysogeny (12).
--(al 0.05M KCI
60 -
40 -
$ 20-:0
0; 0
z (b) 0.5M KCI
AC
0 5 10 15 20 25
Fraction number
mu
FIG. 5 (left). V elocity sedim entation of native Cro protein in a
glycerol gradient. Cro protein (0.2 ml of Fraction V) was layered on a
10 to 30% glycerol gradient in Buffer B containing either 0.05 M KC1(a) or 0.5 M KC1 (5) and sedime nted for 24 h at 49,000 r-pm. The
vertical arrows denote the sedim entation posi tion of marker proteins:B is bovine serum album in (M, = 65,000); 0, ovalbum in (45,000); C,
chymotrypsinogen (25,000); M, myoglob in (17,000). O-O, DNA-
bindin g activity for X-DNA; +O, DNA -binding activity forAimm434-DNA.
FIG. 6 (right). Electro phore sis of denatured Cro protein in a poly-
acrylamide gel containing sodium dodecyl s ulfate. Cro protein (7 pg
of Fraction V) was treated with 1% sodium dodecyl sulfate and 2% 2-mercaptoe thanol for 16 h at room temperature, and electrop horesis
in 10% nolvacrvlamide and 0.1% sodium dodecvl sulfate was carried
out for !P/i h ai 8 mA/tube as described by Weber and Osborn (19).Protein was stained with Coomassie brilliant blue. The molecular
weight w as estimate d from marker proteins (bovine serum album in,ovalbum in, chymotrypsinogen , myoglob in, and cytochrome c) run in
a parallel gel.
We wanted to determine the dissociation constant for Cro and
compare it to the very low value of lo-l3 M estimated for c1
protein.
In order to analyze the binding data, we needed to know
that Cro binding is suff iciently specific for the operator sites on
A-DNA for this interaction to dominate the binding curve and
that one active Cro protein is suff icient to retain the radioac-tive A-DNA on the nitrocellulose filter in the standard binding
assay . This information is provided by the binding curve of
Fig. 7, in which Cro concentration is varied for two DNA
substrates, A and Aimm434; Aimm434 is mainly identical with
A but lacks the region of A-DNA containing the specific bind-
ing sites for c1 and Cro proteins (7, 10, 12). The binding to A-DNA is linear at low concentrations of Cro protein, indicating
that 1 act ive Cro molecule can retain 1 A-DNA mo1ecule.2 The
binding is also largely specif ic for A-DNA. The “nonspecific”
binding that we have observed for Aimm434-DNA has also
been found for other DNA-binding regulatory proteins (26,271,and presumably represents relatively weak binding interac-
tions that can occur anywhere on a DNA molecule (a similar
binding curve to that of Aimm 434 has been found also for (P80
DNA). From the data of Fig. 7, we conclude that the standard
* Because of the relatively large dissociation constant of Cro pro-
tein, the wash ing procedure used for the filter assay is important(see “Methods”).
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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 7 10.0
Tryptophan 0 0.0
Tyrosine 3 3.8
Valine 4 5.1
Total 78
o Calculations were made using the assumption that the protein
has 1 histidine residue per monomer unit.
- -0 20 40 60
Cro concentration, rig/m l
FIG. 7. DNA binding of Cro protein as a function of Cro concen-
tration. Binding assays were carried out using the standard bindingconditions (DNA concentration, 2 pglml), but the filter was washed
with 0 .5 ml of 45% ethanol as describ ed under “Methods.” O-O,
DNA -binding activity for h-DNA; O-O, DNA -binding activity for
himm434-DNA.
DNA-binding assay will allow us to estimate specific binding
constants.
A binding curve in which DNA concentration is varied is
most appropriate for the measurement of an equilibrium disso-
ciation constant (28, 29); the results of this experiment for Croprotein are shown in Fig. 8. The detailed interpretation of the
binding curve is complicated by the fact that we do not knowprecisely how many specific binding sites for Cro are present
on a A-DNA molecule. For A c1 protein, there are three binding
f”operator”) sites on either side of the cI gene, termed ~a,, oa2,
and or,:) and oi,,, oL2, and oL3; of these oR, and oL, have substan-
tially higher affin ities for c1 than the others (30). For Croprotein, we only know so far that Cro binds to at least two s ites
in the 0,. and oH region (see Ref. 12 and below). I f we assume
two binding sites per 3 x lo7 daltons of A-DNA (311, we
calculate a dissociation constant of 8 to 9 x 10-l’ M. Compari-
sons with dissociation constants of other proteins are diff icul t
Of I 1 1 I0 2.5 5.0 7.5 10.0
DNA concentration, pg/m l
FIG. 8. DNA binding of Cro protein as a function of DNA concen-tration. Bind ing assay s were carried out as for Fig. 7, except that h-
DNA concentration was varied as indicated on the figure and Croconce ntration was held consta nt at 15 rig/ml (O---O) or 10 rig/ml
(0-O).
I I t I I I I0 2 4 6
Time, min
FIG. 9. Stabi lity of Cro.h-DNA complex. A-13*P1DN A (0.5 pg/ml)
was incubated with Cro protein for 10 min at 0” at a binding level of
20% of the input DNA (zero time), unlabe led A-DNA was added to 15
pglm l, and O.l-ml ali quots were taken out at intervals and filtered todetermine the remainin g A-[32PlDNA .Cro comple x. O-O, at pH
7.3; O---O, at pH 6.6.
to interpret because the measurements are subject to substan-
tial variation with ionic strength, temperature, and pH. How-
ever, Cro does appear to be a substantially weaker DNA-
binding protein than other specif ic regulatory proteins studied
so far ; approximate values for A c1 protein, lac repressor, andaraC protein are lo-l3 M (7), lo-l3 M (281, and lo-‘* M (29),
respectively.Y
Dissociation Rate Constant - To estimate the dissociation
rate constant, we formed a Cro.X-13’PlDNA complex and mea-
sured the loss of X-1321DNA from this complex with time in thepresence of a 30-fold excess of unlabeled A-DNA (Fig. 9). Since
a Cro molecule should not reassociate to a significant extent
with the 132P1DNA once released, the initial decrease in
[32P]DNA bound should follow an exponential first order decay
:I Since the number of binding sites for Cro is at least two, anunderestim ate of the number of Cro sites will only increase thedifference s between Cro and other spe cific regulatory proteins.
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Cro Protein of Phage A
a$ 25
0 4 8 12
Cro protein, pg/m l
FIG. 10. Repres sion of total RNA sy nthesis by Cro protein. RNA
synthesis was carried out using purified RNA polymerase and hb2 orhb2imm434-DNA as a template as described under “Methods.” The
DNA was first incubated with the levels of Cro protein indicated onthe figure, RNA polymerase was then added, and RNA synthesis
was initiated by the addition of four nucleoside triphosphates to-
gether with rifampicin. Without Cro protein, 16 and 9 pmol of
13HlUMP were incorporated for A- and Aimm434-DNA, respectively,during the incub ation for 10 min at 30”. O--O, RNA synth esis with
A-DNA; O-O, RNA synthesis with Aimm434-DNA.
curve. Because of the rapid decay found under our standard
binding condition of pH 7.3, we also carried out binding assays
at pH 6.6. The calculated dissociation rate constant is about 2
x lo-* s-l at pH 7.3 and 5 x 10m3 s-l at pH 6.6. As expected
from the equilibrium binding data, the half-life for Cro disso-
ciation at pH 7.3 is much less than that found for c1 protein
and lac repressor, and substantially less than the 3-min value
found for araC protein. In an experiment carried out underidentical conditions, we found the half-li fe for c1 dissociation to
be greater than 100 min. We have also attempted to measure
the dissociation rate for “nonspecific” binding, using DNA
lacking the specif ic operator sites (kimm434- or @80-DNA).The dissociation was very rapid with a half-li fe of 510 s, which
is consistent with the concept that the nonspecific binding is asubstantially weaker interaction than the specif ic binding to
the regulatory sites.
We have attempted to determine the association rate con-
stant, but have not been able to do so because Cro protein is
unstable at the very high dilution required for this measure-
ment. If the rate constant for Cro is comparable to that esti-
mated for araC protein (2 x 10y M-’ SK’), the value calculated
for the equilibrium dissociation constant is lo-” M at pH 7.3.
Effect of Cro Protein on RNA Synthesis
From experiments carried out in viv o, we expect Cro protein
to act as a specific repressor of RNA synthes is initiated at theearly promoter sites of A-DNA, pL and pR (see Refs. 1 to 6 and
Fig. 1). To test this expectation, we used purified A-DNA as atemplate for RNA polymerase and studied the eff ect of Cro
protein on RNA synthesis. To ensure that Cro effect s derive
from the specific binding reaction, we used Ximm434-DNA as a
template in parallel experiments.
The capability of Cro protein to function as a specif ic repres-
sor in vitro is shown in Fig. 10, in which strong repression of
total RNA synthes is from A-DNA occurs in the absence of any
repression eff ect on RNA synthesis f rom Aimm434-DNA. The
RNA products were analyzed further by separation throughpolyacrylamide gel electrophoresis and visualization by auto-
8-9S(
6S--,
4s-+ :
a b cFIG. 11. Effect of Cro protein on individual RNA chains. RNA
synthesis was carried out in the presence of p factor using 13*PlUTP .Th e 13*PlRNA was extracted, subjected to electrophoresis in a 3.5%polyacrylamide slab gel containing 8 M urea, and identifi ed byautoradiography as describ ed under “Methods.” a, A-DNA, no Cro
protein; b, A-DNA, Cro protein added at 3 pg/ml; c, Aimm434-DNA,
Cro protein added at 3 pg/ml.
TABLE III
Comvetition between Cro and RNA polvmerase-Proteins and order of addition
L3HlUMP in- Repres-corporated sion
pITlO %
RNA polymerase” 16.5 0
Cro and then RNA polymeraseb 3.2 81
RNA polymerase and Cro togetherc 3.5 79
RNA polymerase and then Crod 16.4 1
a RNA polymerase was incuba ted with A-DNA at 17” for 20 min
before addition of nucleoside triphosphates and rifampicin (see
“Methods”).
* Cro protein was added to A-DNA for 10 min at 17” followe d by an
RNA polymerase incubation as for Line 1.
c Cro protein and RNA polymerase were added to A-DNA together
followe d by an incub ation at 17” for 30 min.
d RNA polymerase was incubated with A-DNA as for Line 1, and
Cro protein was then add ed for 10 min at 17” before additio n of
nucleoside triphosphates.
radiography (Fig. 111. The RNA produced in vitro from A-DNA
in the presence o f p factor is predominantly of four size classes,
designated 4 S, 6 S, 8 to 9 S, and 12 S (14,18,32,33). The 8 to 9
S and 12 S transcripts represent RNA chains initiated at thep,
andp, promoters, respectively, and the 4 S and 6 S transcriptsrepresent promoters near the ~11 and Q genes, respectively(Fig. 1). The results of Fig. 11 show that Cro protein represses
8 to 9 S and 12 S RNA synthes is from A-DNA but not from
Aimm434-DNA; 6 S RNA synthesis was not repressed by Cro
with A-DNA as a template (although not shown at this gel
exposure, 4 S RNA was also not repressed by Crol. As expected
from the failure of Cro to repress total RNA synthesis from
Aimm434-DNA (Fig. lo), there was no difference in the gel
pattern of RNA synthesis f rom Aimm434-DNA in the presence
or absence of Cro (data not shown).If the repression of transcription noted in Fig. 10 and Fig. 11
occurs at the initiation step of RNA synthesis, the binding of
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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 effect ive repressor (Table I II, 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 gif t of termination
probably represses RNA s.ynthesis by blocking the capaci ty of factor p
RNA polymerase to bind at the promoter site; a similar mecha-
nism has been inferred for c1 protein (7-11).From these results, we conclude that Cro is an effect ive and
specif ic repressor which inhibits the initiation of RNA synthe-
sis from the early promoters, pL and pR. Since 8 to 9 S RNA is
the cro gene RNA, Cro protein functions as an “autorepressor”that regulates its own synthesis.
DISCUSSION
Cro protein has a special role among the specific regulatory
proteins analyzed so far because it functions during a temporal%vitch” in viral development from a replication-oriented
“early” stage to a maturation-oriented “late” stage. The
repression act ivi ty of Cro serves to turn down the transcription
of early genes concerned with production of replication and
recombination proteins during the period of head and tailproduction, virus assembly, and cell lys is. The mechanism for
this essential time delay in the action of Cro has been a puzzle
because the cro gene is transcribed during the earliest (“imme-
diate early”) stage of RNA synthesis (l-5). Our biochemicalexperiments suggest that the delayed action of Cro might
result from the relatively low affini ty of Cro protein for itsspecif ic regulatory sites; thus Cro will begin to function as a
repressor only after the time required for the synthesis of a
high level of the protein.
In contrast to Cro, the c1 protein of phage A functions as a
steady state repressor to maintain lysogeny through a com-plete turn-off of transcription of early genes. We suggest that
the different biological requirements for the repression of
early genes during lyti c growth or lysogeny has led to theevolution of biochemically different repressors, a high aff ini ty
maintenance repressor (~1) and a low aff ini ty lyt ic repressor
(Cro), although both use the same operator region of h DNA.
Cro and c1 also appear to diff er in their eff ect on transcriptionof the cro and c1 genes. Because they repress transcription
initiated at pro both c1 and Cro are repressors of the CM gene
(thus Cro is an “autorepressor”); however, c1 can function
either as a repressor or an activator of the c1 gene transcriptinitiated at pM, whereas Cro probably functions only as a
repressor of this RNA (Fig. 1) (l-12, 30). This additional
functional difference between the two repressors should be
clarified by a more detailed analysis of binding and transcrip-
tion.
Cro protein is the smallest DNA-binding regulatory proteinpurified so far that exhibits specificity for a DNA site. Re-markably the amino acid composition of Cro protein is very
similar to that of the transcription facto r TFl of phage SPOl
(351, the prokaryotic DNA-binding protein HU (23, 241, and
the eukaryotic histone H2B (25); the similarity to HU protein
is mosl striking. HU is also a small protein (molecular weight7000) but lacks any known spec ific ity for DNA sites (231. An
interesting possibility is that HU protein might have a mini-mal structure for recognition of double-stranded DNA, to
which Cro protein has added a minimal structure for specif ic
sequence recognition. A comparable structural analysis of
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