volume 17 Number 19 1989
Nucleic Acids Research
The cl repressor of bacteriophage PI operator-repressor interaction of wild-type and mutant
repressor proteins
Jochen Heinrich, Hans-Dieter Riedel, Barbara R.Baumstark', Makoto Kimura+ and Heinz Schuster*
Max-Planck Institut fur Molekulare Genetik, Berlin 33, FRG and 'Department of Biology, Georgia State
University, Atlanta, GA 30303, USA
Received April 10, 1989, Revised July 7, 1989; Accepted August 29, 1989
ABSTRACT
The cl repressor gene of bacteriophage PI and the temperature-sensitive mutants Plcl. 100 and
Plcl.162 was cloned into an expression vector and the repressor proteins were overproduced. A
rapid purification procedure was required for the isolation of the thermolabile repressor proteins.
Identification of the highly purified protein of an apparent molecular weight of 33,000 as the product
of the cl gene was verified by (i) the coincidence of partial amino acid sequences determined
experimentally to that deduced from the cl DNA sequence, and (ii) the temperature-sensitive binding
to the operator DN A of the thermolabile repressor proteins. Analysis of the products of c l - c l . 1 0 0
recombinant DNAs relates the thermolability to an unknown alteration in the C-terminal half of the
cl.100 repressor. Binding to the operator DNA of cl repressor is sensitive to N-ethylmaleimide.
Since the only three cysteine residues are located in the C-terminal half of the repressor it is suggested
that this part of the molecule is important for the binding to the operator DNA. This assumption
is supported by the findings that a 14-kDa C-terminal repressor fragment obtained by cyanogen bromide
cleavage retains DNA binding properties.
INTRODUCTION
The repressor of bacteriophage PI, encoded by the cl gene, is responsible for maintaining
the prophage PI as a plasmid in the lysogenic state. The cl repressor interacts with 14
repressor binding sites or operators on the PI genome (1-5). Recently, the cl gene was
cloned into expression vectors and the repressor protein was overproduced and purified
to near homogeneity (6). Although the DNA binding activity of a protein fraction derived
from a cl temperature-sensitive mutant was found earlier to be thermolabile in vitro (7),
attempts to purify the thermolabile repressor protein have been unsuccessful so far (6).
The isolation and characterization of a cl mutant protein are, however, essential to establish
unequivocally that the PI repressor is the product of the cl gene. Therefore, we have
developed a rapid purification procedure for the isolation of the repressor proteins from
two temperature-sensitive Plcl mutants. This procedure is described here. A partial amino
acid sequence of the repressor proteins, also reported here, is in complete agreement with
the sequence of a 283-codon open reading frame deduced from DNA sequence analysis
of the cl gene (see the accompanying paper by Osborne et al., reference 8).
The Plcl repressor has been shown to recognize a large number of operator sites located
at widely separated regions of the PI genome. DNA sequence analysis of 14 cl-controlled
operators reveals a 17-bp consensus sequence, A|TTGCTCTAATAAATTT|7. The
nucleotides at positions 4, 5, and 7 - 1 0 of the operator play an essential role in repressor
recognition, as judged from the analysis of operator-constitutive mutants of the operator
Op72 of PI (9). The consensus sequence recognized by the Plcl repressor is not symmetric
© IRL Press
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Table 1. Piasmid constructions
Plasmid
Plcl DNA fragment/vector
Reference
pBR325-Pl:7
pBR325-Pl:7.100
pBR325-Pl:7.162
pBR325-Pl:9 (Op51 DNA)
Pl:7 in pBR325 (EcoRQ
Pl:7.100 in pBR325 (EcoW)
Pl:7.162 in pBR325 (EcoRI)
Pl:9 in pBR325 (EcoRI)
(3)
this paper
this paper
(3)
pBD2
[PwWBcd] subfragment of Pl:7
in pPLc28 [EcoRl/BamHl]
[BaWBdl] subfragment of Pl:7
this paper (Fig.l)
inpJF118EH [SmallBamWl]
same as pMV2w except PI:7.100
same as pMV2w except PI :7.162
cl control region Op99 a to e
in pUC19 [SmaVHincU]
pMV2w
pMV2.10O
pMV2.162
pMT307 (Op99 DNA)
(6)
this paper
this paper
this paper (Fig. 1)°
Linearization of the vector DNA was done with the restriction enzyme in parentheses. Brackets indicate that
the enzyme recognition site is lost in the recombinant DNA.
*the cl control region Op99 a to e is 600 bp long with the Pvull site at the left end (Velleman, M., and
Heinzel, T., personal communication).
and hence has a directionality (1—5). In this respect, cl repressor binding differs from
the binding of several well-characterized repressor proteins (10), and resembles instead
a separate class of regulatory proteins, which include the products of the PlrepA gene
(11), the lambda cU gene (12), and a repressor gene of </>105 (13). In addition, the amino
acid sequence of the Plcl repressor reported here and deduced from the DNA sequence
(8) does not reveal a helix-tum-helix motif that has been associated with DNA binding
proteins which recognize operators with a two-fold symmetry (10). Therefore, we have
looked for other domains of the cl repressor which may be important for the binding to
the operator. We report that the binding ability is lost upon treatment of the repressor
with N-ethylmaleimide. Since the only three cysteine residues are located in the C-terminal
half of the repressor, we suggest that this part of the molecule plays an active role in the
binding reaction.
MATERIALS AND METHODS
Bacterial and phage strains
The following E.coli strains were used: C600, HBlOlrecA, GM82dam, and K140. Plclts
phage were obtained by heat induction of C600(PlCmO cl. 100) (14) and K140 (Plcl. 162)
(15). PI DNA was obtained from Plclts phage by phenol extraction as described (16).
Recombinant plasmids
The construction of recombinant plasmids containing the vector pBR325, pPLc28, and
pJFl 18EH (17) was done essentially as described (6) and is summarized in Table 1. To
begin with, PI EcoRl fragments were inserted into £coRI-linearized pBR325. The Plcl
gene-containing plasmids were propagated in strain GM82. From the plasmid DNA a 2.3
kb Bell subfragment containing the cl gene was then isolated. A subsequent treatment
with Ball separates the cl gene from its control region Op99 (Fig. 1). The BaJl-BcR
subfragment containing the cl gene was then inserted into the Wc-controlled expression
vector pJF118EH (Table 1 and Fig.l).
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Op99
* * .
d gene
d
c
a
I I* •
•
•—H
Ba/1
Ben
Bg/ll
Op99-DNA
EcoRI
pMV2w
Hindlll
[Ba/1]
tac H
[Bcfl]
I
I
pMV2w/100
pMV2.100/w
pMV2.100
EcoRI
H/ndlll
Fig. 1. Subcloning of the cl gene from PI wild-type and mutant rcpressor strains. The 2.3-kb Bc/I subfragment
of Pl:7 containing the cl gene (open bar) and the cl-control region Op99 is shown in the upper part. Op99
contains four repressor binding sites or operators, Op99 a to e (5). In the Op99 DNA the control region is inserted
into pUC19 DNA (for the construction see Table 1). The EcoRI and Hindlll sites of the pUC19 polylinker region
are indicated by triangles. TheBa/1-BdI subfragments of PI wild-type and Plcl. 100 DNA inserted into pJF118EH
(toe) DNA are shown in the lower part. Recombinants of PI and Plcl. 100 DNA (white and black bars, respectively)
were constructed by exchanging the corresponding Bg[U-HindIH subfragments of pMV2w and pMV2.100 DNA.
(The EcoRI and HindQi sites are in the polylinker region of pJFl 18EH and are indicated by triangles).
Proteins and enzymes
Plcl wild-type repressor was induced in HB101(pMV2w) (Fig.l) by IPTG. Plcl*, a
primary proteolytic degradation product of cl repressor, was isolated from
C600(pBD2,pcI857) after heat induction of the cl repressor (6). Both the cl and cl*
repressor were purified as described previously (3,6). Restriction endonucleases, T4 DNA
ligase, egg white lysozyme, and aprotinin were from Boehringer and were handled as
described by the manufacturers and by Maniatis et al. (18). The low molecular weight
protein markers were from BioRad. Trypsin was from Merck.
Chemicals
The following chemicals were used: cyanogen bromide (Fluka), N-ethylmaleimide (Sigma),
heparin-Sepharose CL-6B and DEAE-Sephacel (Pharmacia), CM52-cellulose (Whatman).
Media and buffers
Bacteria were grown in TY medium (16). Buffers used were Buffer A: 20 mM Tris-HCl
(pH 7.6), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 10% (v/v) glycerol; Buffer B:
50 mM Tris-acetate (pH 6.8) 10% (v/v) glycerol; Buffer C: 20 mM K-phosphate (pH 7.0),
50 mM NaCl, 0.1 mM EDTA, 10% (v/v) glycerol; Buffer D: 20 mM K-phosphate (pH
7.0), 50 mM NaCl, 1 mM EDTA, 1 mM DTT; Buffer E: same as Buffer A + 0.1 mg/ml
BSA.
cl repressor assay
DNA mobility shift. Binding of repressor to the operator was monitored as described
previously (3,6). If not otherwise noted, 50 to 250 ng of repressor and 0.2 to 1 /tg of operator
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[kDa]
a
b
c
d
e
cl c1 ifeaioo
92,5
66,2
45
31
21.5
14.4
Fig.2. Purification of cl repressor proteins. Left: purification of cl. 100 repressor. lane a: Markers (2 /jg each)
are in descending order phosphorylase B, BSA, ovalbumin, carbonic anhydrase, soybean trypsin-inhibitor, and
lysozyme; lane b - c : crude extract (30 /J each) of C600(pMV2.100) before (b) and after (c) induction; lane
d - e : Fraction II (20 /J) and Fraction III (20 iii), respectively. Right: the purified repressor proteins cl, cl*,
cl.162, and cl.100 (2/tg each) from left to right. The probes were subjected to 17.5% SDS-PAGE. Note, that
cl.100 repressor-containing fractions contain BSA as described.
1
51
MINYVYGEQLYQEFVSFRDLFLKKAVARAQHVDAASDGRPVRPVWLPFK
A
S(ci.ioo)
ETDSIQAEIDKWTIJIARELEOYPDLNIPKTILYPVPNILRGVRKVTTYQT
101
EAVNSVKMTAGRIIHLIDKDIRIQKSAGINEHSAKYIENLEATKEmKOY
151
PEDEKFRMRVHGFSETMLRVHYISSSPKYNDGKSVSYHVLLCGVFICDEt
A
A
A
A
A
201
LRDGIIINGEFEKAKFSLYPSIEPIICDRWPQAKIYKLADIENVKKOIAI
251
TREEKKVKSAASVTRSRWKKGQPVWPNPESAQ
Fig. 3. Amino acid sequence of the cl repressor. The amino acid sequence which is deduced from the 283-codon
open reading frame of cl is verified by the determination of partial sequences of tryptic digestion products from
cl.100 (underlined sequences) and cl* repressor. The peptide(s) which is absent in cl* repressor is represented
by an interrupted line and the possible C-termini of cl* are indicated by vertical arrows. At the position 55 of
the cl. 100 repressor isoleucine is replaced by serine. The positions of methionine residues are pointed out by
triangles. Note, that 3 cysteine residues are present at the positions 192, 197, and 227, respectively.
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DNA were incubated in buffer E (25 ftl total volume) for 15 min at 37 °C (cl wild-type
repressor) or 30°C (cl. 100- and cl. 162 repressor). Double-digested pMT3O7 DNA (EcoRl
+ Hindm) and pBR325-Pl:9 DNA (£coRI + BstnT) were used as operator DNA and
for convenience are abbreviated Op99 DNA and Op51 DNA, respectively.
Lysogenization with PICmO cl. 100. The thermoresistance of cl repressor activity in vivo
was tested by infection of bacteria with PICmO cl. 100 and screening for thermoresistant
Cmr colonies as described (6).
Amino acid sequence analysis
N-terminal sequences of the protein and peptides were determined by the 4-N,NdirrKmylaminoazobenzene-4'-isothiocyanate/phenylisothiocyanate (DABrTC/PITC) doublecoupling method (19). Tryptic digestion was carried out as described (20). Lyophilised
protein (~ lmg) was dissolved in 0.4 ml 0.2 M N-methylmorpholine acetate buffer (pH
8.1). Trypsin was added at 1/50 (w/w) ratio, and the mixture was incubated at 37°C for
4 h. The digest was lyophilised, dissolved in 100 /il 0.1 % trifluoroacetic acid, and purified
by reverse-phase high-performance liquid chromatography (RP-HPLC) on a column of
Vydac C18 using an acetonitrile gradient in aqueous trifluoro acetic acid. The effluents
were monitored by absorption at 220 run.
RESULTS
The construction of cl-gene containing recombinant plasmids for the overproduction of
cl repressor is described in Fig. 1 and Table 1. In essence, it follows the procedures
described previously (6). For the purification to near homogeneity of cl wild-type repressor,
cellular crude extracts were treated with streptomycin sulfate, the proteins were precipitated
with ammonium sulfate and subjected to heparin-Sepharose-, DEAE-Sephacel- and CMSepharose chromatography in that order (6). When the same procedure was applied for
the isolation of the cl. 100 repressor, the latter could not be recovered from heparinSepharose (6). Therefore we developed a rapid isolation method for thermolabile cl
repressor proteins which is described here.
Purification of cl mutant repressor proteins
cl.100 repressor. A culture (400 ml) of C600(pMV2.100) was grown at 30°C to a
concentration of about 2 x 108 bacteria/ml. Repressor synthesis was then induced by the
addition of 1 mM IPTG (final concentration). Following an incubation for 2 hours at 30°C
the bacteria were harvested by centrifugation [5,000 rpm (GS3) 5 min, 2°C] and the pellet
(3 g of wet cell paste) was resuspended in 12 ml 200 mM NaCl, 20 mM spermidine,
and 2.5 mM EDTA. Bacteria were lysed with 0.5 mg/ml lysozyme in a lysis mixture
which contained 25 mM TrisHCl (pH 8.0), 180 mM NaCl, 2 mM EDTA, 15 mM
spermidine, 3.5% (w/v) sucrose, and 0.1% (w/v) Brij 58 (final concentrations). After
incubation for 30 min at 0°C to - 2 ° C bacterial lysis was completed by warming up of
the suspension to 10°C (2 min) with stirring. The clear lysate was adjusted to 750 mM
NaCl and centrifuged [35,000 rpm (45Ti), 50 min, 2°C]. The supernatant was diluted
5-fold with Buffer A without NaCl (Fraction I, 85 ml). Fraction I was mixed with 5 ml
of heparin-Sepharose which had been equilibrated with buffer A, and the mixture was
gently moved at 0°C for 30 min. After centrifugation [5,000 rpm (GSA), 5 min, 2°C]
the sediment was resuspended in buffer A and filled into a column. The column was washed
with 20 ml buffer A + 150 mM NaCl. After applying a 50-ml linear gradient of 150
to 1000 mM NaCl in buffer A the proteins were eluted from the column into tubes containing
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10/il of 10 mg/ml BSA each. 1-ml fractions were collected and the repressor-containing
fractions were pooled and dialyzed against buffer B (Fraction n, 6 ml). Fraction II was
applied to a 2-rnl DEAE-Sephacel column and the repressor-containing flow-through was
loaded directly onto a 1.2-ml CM-Cellulose column. Both columns were washed with buffer
B and the repressor was eluted from the CM-Cellulose with a 12-ml linear gradient of
50 to 600 mM Tris-acetate (pH 6.5) in buffer B. 200 /il-fractions were collected in the
presence of 0.1 mg BSA/ml (final concentration). Repressor-containing fractions were
pooled, dialyzed against buffer A for 3 hours, diluted 1:2 with 87% (v/v) glycerol and
kept at -70°C (Fraction m , 2 ml).
Starting with bacterial lysis, the whole procedure lasted about 30 hours. In order to
preserve the operator binding capacity of the cl.100 repressor, ammonium sulfate
precipitation and freezing of protein fractions had to be avoided. For the same reason BSA
had always to be present at later purification stages.
c 1.162 repressor. The construction of a recombinant plasmid containing the repressor gene
of Pic 1.162 DNA was done in exactly the same manner as described for the cl wild-type
gene (Fig.l). Therefore, plasmid pMV2.162 is identical to pMV2w (Fig. 1) except of the
c 1.162 mutation. Repressor synthesis in HB101(pMV2.162) bacteria was induced with
IPTG and the repressor protein was purified as described above for the cl. 100 repressor.
The cl.162 repressor appeared to be more stable than the cl.100 repressor. During the
purification procedure the proteins could be concentrated by ammonium sulfate (0.42 g/ml)
precipitation and BSA could be omitted without affecting the operator binding capacity
of the cl. 162 repressor. The purification of the repressor proteins was monitored by SDSPAGE. As shown in Fig. 2, the purified mutant repressor proteins comigrate with the
purified cl repressor polypeptide at 33,000 Mr.
cl*, a polypeptide of 31,000 Mr (Fig.2), was shown previously to be a truncated
derivative of the 33,000 Mr cl repressor (3). Although cl* is missing 6 - 8 % of the cl
amino acid sequence, its DNA binding properties are indistinguishable in vitro from those
of the wild-type protein (6), suggesting that the missing amino acids are not essential for
DNA binding specificity.
Amino acid sequence analysis of the cl repressor
To determine whether the apparently nonessential amino acids missing from the cl*
repressor are normally located at the N-terminal or the C-terminal part of the cl protein,
we carried out amino acid sequence analysis of the cl* repressor and compared the results
with the analysis of a full-length repressor protein (cl. 100 repressor). N-terminal sequencing
of both the cl* repressor and the cl.100 repressor by the DABITC/PITC method gave
the sequence Met-De-Asn-Tyr, suggesting that the amino acids lacking in the cl • repressor
are derived from the C-terminal region of the protein. Furthermore, the sequence MetDe-Asn-Tyr corresponds to the start of an open reading frame (ATGATAAATTAT) located
within the region predicted to contain the beginning of the cl gene (2, 8). This open reading
frame extends for 283 amino acids downstream of the Met-De-Asn-Tyr coding sequence
(8), yielding a polypeptide of Mr 32,512, a value that corresponds well with the molecular
weight predicted by SDS-PAGE of the purified repressor proteins (Fig.2). To determine
whether cl* is truncated at the C-terminal end, cl* and cl. 100 were subjected to tryptic
digestion and the peptides were separated by RP-HPLC as described in Materials and
Methods. A comparison of elution patterns of the peptides showed that the two C-terminal
peptides Thr-Lys and Lys-Gly-Gln-Pro-Val-Asn-Asp-Asn-Pro-Glu-Ser-Ala-Gln are missing
in the cl* repressor (Fig.3). Although it is not possible to determine unambiguously the
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Table 2. Plcl recombinant plasmids promote lysogenization by PICmO cl.100
Plasmid
40°/30°C ratio of Cm' colonies
pMV2.w
pMV2.w/100
pMV2.100/w
pMV2.100
5,6
<3xlO~5
4,2
<8xlO~6
The structure of the plasmids is shown in Fig. 1. Plasmid-containing C600 bacteria were infected with PICmO
cl.100 (moi = 8, 15 min, 30°C) and serial dilutions of the infected bacteria (25 /J) were applied in duplicates
to agar plates + 25 ftg/ml chloramphenicol. A series of plates was incubated at 30°C the other at 40°C. Cm'
colonies were counted after overnight incubation.
C-terminal residue of cl* because of the clustering of basic residues, the results suggest
that the C-terminal 14 to 16 amino acid residues of the repressor molecule are absent in
cl*. The loss of these amino acids roughly corresponds to the decrease in the molecular
weight of cl* versus cl repressor (Fig.2).
Within the regions sequenced, the cl.100 repressor differs from the cl* repressor at
a single position 55 residues from the N-terminal end. At this position, the cl. 100 repressor
contains a serine, while the cl* protein contains an isoleucine. Isoleucine is also the amino
acid predicted to occur at this position by DNA sequence analysis of the wild-type cl gene
(2, 8). To determine whether the Ile-Ser substitution at residue #55 is responsible for
the thermolability of the cl.100 repressor, recombinant DNAs were constructed in which
the C-terminal parts of the repressor genes (the BglH-HindHl fragments, Fig.l) were
exchanged between cl wild-type and cl.100. The recombinant repressor protein encoded
by these plasmids was then tested for its thermostability. As can be seen in Table 2, the
repressor encoded by pMV2.100/w (which contains the cl. 100 mutation at position # 55)
is not thermolabile, while the repressor encoded by pMV2w/100 (which contains the Cterminal portion of the cl.100 gene) retains the temperature sensitivity characteristic of
the cl.100 protein. These observations localize the cl.100 mutation responsible for
C1.162
C1.100
Op51
Fig. 4. Operator-binding of cl repressor proteins: dependence on temperature, cl, cl.100, and cl. 162 repressor
(50 ng each) was incubated in buffer D for 3 min at the indicated temperatures. After addition of 400 ng of
Op51-DNA the samples were incubated for 15 min at the same temperature and subsequently subjected to 1.2%
agarose gel electrophoresis. Op51, the DNA fragment containing the operator Op51; Left lane, 400 ng of
Op51-DNA.
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Ci.100-Op99
25 30 35 40 45 [ C]
Op99
Fig. 5. Temperature-sensitivity of free and operator-bound mutant cl repressor. A. cl.100: 250 ng of cl.100
repressor was incubated in buffer E for 15 min at the indicated temperatures. 1 y.% of Op99 DNA was then added
and incubation was continued for 15 min at room temperature. cl.l00-Op99: 250 ng of cl.100 repressor and
1 /ig of Op99 DNA were incubated for 15 min at room temperature. After a shift to the indicated temperatures,
incubation was continued for 15 min at the same temperature. B. c 1.162 and cl.l62-Op99: The same procedure
was used as described under A except that 200 ng of c 1.162 repressor was used. Op99, the DNA fragment containing
the operators Op99a, c, d, and e. X HindUl- and 0X174 HaeVA DNA fragments (1 /ig each) were used as marker
(M). Second lane in A and B, 0,4/ig of Op99 DNA. The probes were subjected to 1.5% agarose gel electrophoresis.
thermolability to a region within the open reading frame that is distal to the BglU. site,
and thus at least 63 amino acids from the N-terminus (Fig.3).
Temperature-sensitivity of cl mutant repressor proteins
The temperature-sensitivity of the cl.100 and cl.162 repressor was tested in two
mMNEM
Op99
Fig. 6. NEM-sensitivity of free and operator-bound cl repressor. cl: samples of cl repressor (50 ng each) were
incubated in buffer C for 15 min at 37°C at NEM concentrations as indicated. Op99 DNA (1 jig each) was
then added and incubation was continued for 15 min at 37°C. cl-Op99: cl repressor and Op99 DNA (as above)
were incubated for 15 min at 37°C Following the addition of NEM at concentrations as indicated, incubation
was continued for 30 min at 37°C. Op99, as indicated in the legend to Fig. 5; Left lane, 1 ^g of Op99 DNA.
The probes were subjected to 1.2% agarose gel electrophoresis.
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[kDa]
a
92.5
66.2
—
••»
45
31
b
c
d
e
a
b
c
d
«M
«^IZr:
Op51
21,5 ^ ^
™ "
14,4 ^ ^ ^
6.5
fl^
Fig. 7. Isolation and DNA binding of a t e r m i n a l 14-kDa cl repressor fragment. Left: Isolation of the 14-kDa
fragment. Lane a, protein markers as described in the legend to Fig. 2; lane b, 2 jig cl repressor; lane c—e,
14-kDa fragment Fraction I (c), Fraction II (d), and Fraction III (e), respectively. The proteins were subjected
to 19.5% SDS-PAGE. Right: DNA binding of the 14-kDa fragment. Lane a, X HindlU- and *X174 HaeUl DNA
fragments (1/tg each); lane b - c , 300 ng of Op51 DNA without (b) and with 50 ng of cl repressor (c); lane
d - e , 300 ng of Op51 DNA without (d) and with (e) 200 ng of 14-kDa fragment. Op51, the DNA fragment
containing the operator Op51. The probes were subjected to 1.2% agarose gel electrophoresis.
experiments. 1. The repressor was preincubated at temperatures ranging from 25° to 45 °C
and the binding to Op51 DNA was then measured at the same temperature. As can be
seen in the DNA mobility shift assay (Fig.4) binding to Op51 DNA of both thermolabile
repressors is abolished at 40° to 45°C whereas binding of the cl wild-type repressor is
still unaffected at 45°C. (Routinely, binding to operator DNA of cl wild-type repressor
had been measured before at 37°C and 47°C, respectively (3,6)). Binding of cl and cl.162
repressor is specific, since only the mobility of the DNA fragment containing the operator
Op51 is shifted. However, in the presence of cl.100 repressor the mobility of other
fragments besides Op51 is retarded (Fig.4). This loss of the binding specificity may be
due to a partial denaturation of the molecule even at low temperatures. 2. The repressor
was first bound to Op99 DNA at low temperature and the stability of the operator-repressor
complex was then tested at different temperatures. Since the Op99 DNA contains several
operators (Fig. 1), several fragments appear the mobility of which is retarded by repressor
(Fig.5). Again, preincubation at 40°C to 45°C of the thermolabile repressor molecules
alone abolished the subsequent binding to the Op99 DNA. However, when the repressor
is first bound to Op99 DNA the protein becomes more heat-stable. A temperature of 45 °C
is needed to partially release the repressor from its operators.
N-ethylmaleimide-sensitivity of the cl repressor
The cl repressor contains three cysteine residues which are clustered in the C-terminal
half of the molecule (Fig.2). Therefore, we tested as to whether NEM would affect the
DNA binding of the repressor. As shown in Fig.6, binding to Op99 DNA of the repressor
is already completely abolished upon preincubation in 0.1 mM NEM. However, when
the repressor is first bound to the Op99 DNA, it is not released upon treatment with NEM
at concentrations up to 1 mM. A partial release from the DNA is only observed after
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incubation in 10 mM NEM (Fig.6). Obviously, cysteine residues play a role in the repressor
activity.
Isolation and DNA binding of a C-terminal cl repressor fragment
The cl repressor contains six methionine residues which are clustered in the N-terminal
half of the molecule. Therefore, cleavage of the repressor with cyanogen bromide (21)
which occurs at carboxyl termini of methionine, should yield the 116-amino acid long
C-terminus of the repressor as the largest fragment (Fig.2). The isolation of this fragment,
which also contains the three cysteine residues of the molecule, is described here.
6.6 mg cl repressor was dissolved in 70% (v/v) formic acid and treated with 23.5 mg
cyanogen bromide (1.5 ml total volume) in the dark under nitrogen at 25°C for 90 hours.
The clear solution was then concentrated by vacuum evaporation and dialyzed against
distilled water (Fraction I, 1.5 ml). Fraction I was adjusted to buffer D and incubated
for 2 hours at 0°C. Insoluble material was removed by centrifugation [10,000 rpm (SS34),
10 min, 0°C]. The supernatant (Fraction II, 1.5 ml) was loaded onto a 0.6-ml heparinSepharose column. The column was washed with 2 ml Buffer D and the proteins were
eluted with a 5-ml linear gradient of 0.05 to 1 M NaCl in Buffer D. A 14-kDa polypeptide
eluted at about 0.5 M NaCl (Fig.7, left). Fractions containing this polypeptide were dialyzed
against distilled water (Fraction EQ, 1 ml).
N-terminal sequencing of the 14-kDa polypeptide by the DABITC/PITC method gave
leu-arg-val-his-tyr in accordance with the N-terminal amino acids of the largest cyanogen
bromide-fragment (Fig.2). When the cl* repressor was treated with cyanogen bromide
a minor amount of an 11-kDa- instead of a 14-kDa polypeptide was recovered (data not
shown). This result confirms that (i) the 14-kDa polypeptide originates from the C-terminus
of the repressor and (ii) C-terminal amino acids are absent in the cl* repressor molecule.
When the 14-kDa polypeptide is incubated with Op51 DNA, the operator-containing
fragment is not retarded as a discrete band but appears as a smear instead (Fig.7). Since
the appearance and/or mobility of the other DNA fragments is not affected by the presence
of the 14-kDa polypeptide the latter must have retained some specificity with regard to
its DNA binding properties. The 14-kDa polypeptide did, however, no longer interact
with the Op99 DNA as measured by mobility shift experiments (data not shown). The
reason for that failure is not known.
DISCUSSION
Previously, we reported the purification and in vitro characterization of the cl repressor
of bacteriophage PI (6,7). However, the purification procedures described at that time
could not be applied to proteins isolated from temperature-sensitive cl mutants because
the mutant proteins were either rapidly degraded or were inactivated during the isolation
process (unpublished observations). Therefore, we developed a rapid procedure which
allows the isolation of the repressor proteins of Plcl.100 and Plcl.162 in a functionally
active form. Binding to operators in vitro by either mutant protein is specific and is
temperature sensitive when compared to the cl wild-type protein. These findings confirm
the identity of the 33 kDa protein as the product of the cl gene. Furthermore, the Nterminal sequence analysis of the cl.100 and the cl* protein establishes the location of
the start of the cl open reading frame. The molecular weight predicted for the protein
coded by this open reading frame (283 amino acids, or 32,512 daltons) is very close to
the molecular weight (33 kDa) calculated from SDS-PAGE of the purified repressor (Fig.2).
Cleavage of the lambda repressor into two relatively stable fragments with papain has
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facilitated the localization of the DNA binding domain of that molecule (22). Such a
favourable situation does not seem to exist for the PI repressor. Digestion with either papain,
trypsin, chymotrypsin or V8 protease does not yield stable intermediates useful for a
corresponding analysis (unpublished observations). However, our observation that the cl*
protein, although missing 14 to 16 amino acids from the C-terminus of the molecule, is
still able to bind to the operator (3), suggests that the extreme C-terminal region of the
repressor is dispensible for specific DNA binding. On the other hand, it is also unlikely
that the N-terminal region of the repressor is solely responsible for DNA binding. This
follows from our finding that a 14 kDa C-terminal polypeptide retains some DNA binding
capacity, although it is not yet known whether this property is due to the specific interaction
of the protein with the operator. Furthermore, the fact that the binding is sensitive to Nethylmaleimide and the only three cysteine residues are located in the C-terminal half of
the repressor implicates the C-terminal region as an essential determinant of repressor
function.
To date, the cl repressor of bacteriophage PI has been shown to bind to at least 14
operator sites located at widely separated positions on the PI genome. In this respect,
the action of the Plcl repressor protein is distinct from that of the lambda cl repressor
and many other well-characterized phage repressor proteins, which tend to bind close to
their own structural gene. The Plcl repressor is also unusual in its recognition of an
asymmetric nucleotide sequence. Thus, as the results reported here suggest, the amino
acid sequence requirements for the DNA binding activity of the Plcl gene product are
likely to differ significantly from those of other phage-encoded repressor proteins.
ACKNOWLEDGEMENTS
We are obliged to S. Iida for the PI phage mutant. The expert technical assistance of A.K. Seefluth is gratefully acknowledged. We thank D. Vogt for the preparation of plasmid
DNA. B. R. B. acknowledges support from the National Sciences Foundation
(PCM-87OU46).
Abbreviations: Pl:7 and Pl:9, PI £coRI fragments nos. 7 and 9; Op, operator; IPTG,
isopropylthiogalactoside; NEM, N-ethylmaleimide; BSA, bovine serum albumin;
*To whom correspondence should be addressed
"•"Present address: Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812,
Japan
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