Cro Regulatory Protein Spe cified by Bacteriophage X STRUCTURE, DNA-BINDI NG, AND REPRESSION OF RNA SYNTHESIS* (Received for publication, February 1, 1977) YOSHINORI TAKEDA, ATIS FOLKMANIS,$ AND HARRISON ECHOLS From the Department of Molec ular Biology, Univ ersit y of California, Berkeley, California 9472 0 The Cro protein spec ified by bacteriophage A is a repressor of the genes expressed early in phag e development and is required for a normal late stage of lyti c growth. We have purified Cro protein to virtual homogeneity and analyzed its struct ure and properties as a DNA-binding protein and re- pressor of RNA synthes is. To confirm that the protein is the product of the cro gene, we have also shown that a missense mutation in the ero g ene leads to a product that is more temperatur e- and salt-sensiti ve in its DNA-bi nding property. As purified, Cro protein is a dimer o f identical subunits of molecular weight 8600. The purifi ed protein binds to A-DNA carrying the specif ic bindi ng sites (operators oL and oli) with an estimated dissociation constant of lo-‘” M to lo-” M; there is also w eake r bindi ng to other si tes on DNA, as found for other DNA-bin ding regulatory proteins. In a purified tran- scription sys tem, the Cro protein is an eff ect ive and speci fic repressor of RNA sy nthesi s from the N and cro genes; thus Cro is an autorepres sor which regulates its own synthe sis. A comparison of the propertie s of the two A repressor proteins, CI and Cro, indicates that cI is a “strong repressor” special- ize d for complete turn off of lyti c functions need ed for the maintenance of lysog eny, wher eas Cro is a “weak repressor ” specialized for a gradual turn off of early viral genes that potentiates the late stage of lyt ic development. The temperate bacteriophage A spec ifie s two repressor pro- teins, c1 and Cro, which carry out regulatory function s essen- tial fo r different aspects o f the viral life cycle. Th e c1 prot ein acts unde r conditions of stable lysogeny to maintain repressi on of the integrate d viral DNA; Cro protein acts durin g lyt ic development to turn of f the exp ression of the phag e genes activ e 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 bindi ng to speci fic operat or sites on X-DNA and ability to repress RNA syn thesis initiated at the X promoter sites activ e early during viral development (7-11 ) (Fig. 1). In a previo us p aper, 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 fro m the National Institute of Gene ral Medic al Sciences. $ Postdoctoral Fellow of the Natio nal Cancer Institute. binding act ivi ty, and establishes the capaci ty of Cro to func- early promoter sites of A-DNA. Our biochemical results indi- cate that the physiological differe nces between c1 and Cro may be attributable to the diffe rent binding capacities of the two proteins. EXPERIMENTAL PROCEDURES Materials Nucleic Acids - Bacteriophage DNA was prepared fr om purifie d phag e by pheno l extraction as describe d previou sly (10, 12). “Chicken blood” DNA and Escherichia coli tRNA were obtained from Calbiochem. Proteins-E. coli RNA po lymera se was prepared as described b y Burgess and Jendrisak (13). Termination factor p, purified according to Roberts (141, was the gif t of J. Gallu ppi and J. Richards on, Universi ty of Indiana. Pancreatic DNase was obtained fro m Worth- ington and ovalbumin, chymo trypsi n, myoglobin, and bovine s erum 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, in which the cro- mutation was tof2 (15). The A mutations and the rationale for using them have been described in more detail prev i- ously (1 2). 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. 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 prepar ed as describ ed by Alberts and Herrick (16). Ultrapure ammon ium sulfate was obtained from Schwarz/Mann, acrylamide and sodium dodecyl sulfate from Bio-Rad, unlabeled nucleoside triphosphates from Sigma, [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 measures the 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 from other h-DNA-binding proteins, we did parallel assays with h-DNA and Aimm434-DNA (which is the same as A-DNA except for the operator-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 DT A. After in cuba- tion for 10 min at O”, the mixture was filtered with a nitrocellulose 6177 byo De ce m be r13, 2008w w w . jbc. o rg Do w lo a de dfro m
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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
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”).
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.
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
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
1.2.3.
4.5.
6.
7.
8.
9.
IO.
11. Maniatis. T.. and Ptashne. M. (1973)Proc. Natl. Acad. Sci. U. S.
12.
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