From the Department of Biochemistry and Molecular Biology, Penn State University College of Medicine, Hershey, Pennsylvania 17033
Received for publication, October 4, 2000, and in revised form, December 24, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sedimentation equilibrium studies show that the
Escherichia coli cyclic AMP receptor protein (CAP)
and lactose repressor associate to form a 2:1 complex in
vitro. This is, to our knowledge, the first demonstration of a
direct interaction of these proteins in the absence of DNA. No 1:1
complex was detected over a wide range of CAP concentrations,
suggesting that binding is highly cooperative. Complex formation is
stimulated by cAMP, with a net uptake of 1 equivalent of cAMP per
molecule of CAP bound. Substitution of the dimeric lacI-18 mutant
repressor for tetrameric wild-type repressor completely eliminates
detectable binding. We therefore propose that CAP binds the cleft
between dimeric units in the repressor tetramer.
CAP-lac repressor interactions may play important roles in regulatory events that take place at overlapping CAP and
repressor binding sites in the lactose promoter.
The Escherichia coli lactose (lac) operon
has long served as a paradigm of bacterial transcription regulation and
is the source of protein-protein and protein-DNA interactions that are
useful as models of transcription-regulatory processes in
Archaea and Eukarya as well. The lac
promoter region contains an array of binding sites positioned to allow
bound proteins to interact in limited and specific ways. Within a
120-base pair sequence are located two binding sites for the
lac repressor, two for
CAP,1 and two for RNA
polymerase (Fig. 1). Proteins occupying
these sites can affect the affinities of other proteins for neighboring sites and can influence, via protein- or DNA-allostery, the catalytic activities of RNA polymerase. For example, under some in
vitro conditions, lac repressor bound to operator 1 stimulates the binding of RNA polymerase to P1 (1) but appears to
inhibit promoter clearance and thus, mature RNA synthesis (2). Under
other higher [salt] solution conditions, the binding of
lac repressor and RNA polymerase in the P1-operator 1 region
appears to be mutually exclusive (Ref. 3 and references cited
therein).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (10K):
[in a new window]
Fig. 1.
Map of the lactose promoter binding sites of
CAP, lac repressor, and RNA polymerase. Data from
Majors (28), Galas and Schmitz (29), Schmitz (30), and Malan and
McClure (31). The repressor binding sites are designated operator 1 and
operator 3, respectively. The starting points for transcripts from
promoters P1 and P2 are indicated by the arrows. To improve
clarity, the P2 binding site of RNA polymerase, located 22 base pairs
upstream from P1, is offset vertically. The scale gives residue numbers
with respect to the start of transcription of promoter P1.
Little is known about the interactions of lac repressor and
CAP in this regulatory system. The DNA surfaces occupied by CAP and
repressor in the CAP site 2/operator 1 region of the promoter are
coincident, whereas those in the CAP site 1/operator 3 region overlap
significantly (see Fig. 1). In vitro, CAP bound at CAP site
1 interacts cooperatively with repressor at operator 1 (4, 5) and
alters the DNase I protection pattern of repressor bound at operator 3 (6), whereas CAP at CAP site 2 is displaced when lac
repressor binds operator 1.2
Although this evidence supports regulatory models in which CAP and
lac repressor interact, it does not allow distinction
between models in which the proteins interact directly, and ones in
which interactions are indirect (mediated, for example, by DNA
conformation change). In addition, it does not address the possibility
that CAP and lac repressor interact when they are not DNA
bound. To determine whether lac repressor and CAP can
interact directly, we have performed equilibrium analytical
ultracentrifugation over wide ranges of [CAP] and [lac
repressor] and the physiological range of [cAMP]. Our results show
that CAP binds lac repressor in a
[cAMP]-dependent manner and that DNA is not required for the interaction.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proteins--
CAP was prepared as previously described (5, 7).
It was homogeneous as judged by SDS-PAGE, 45% active in
cAMP-dependent binding to lac promoter CAP site
1 and >80% active in nonspecific DNA binding. Samples of two
preparations of wild-type lac repressor and two preparations
of lacI-18 mutant repressor were kindly provided Dr. Kathleen Matthews.
The wild-type repressor was >95% pure as judged by SDS-PAGE and
>50% active in lac operator binding. The lacI-18 mutant
repressor was homogeneous by SDS-PAGE and was ~30% active in
lac operator binding (5, 8). CAP and lac
repressor concentrations were determined spectrophotometrically using
CAP 280 nm= 3.5 × 104
M
1 cm
1 (9) and
repressor subunit 280 nm = 2.2 × 104
M
1 cm
1 (10).
Sedimentation Equilibrium Assays--
Samples were brought to
dialysis equilibrium with 10 mM Tris, pH 7.8 at
4 °C, 150 mM KCl, 5 mM MgCl2, 10 µM dithiothreitol, 1 µM leupeptin,
supplemented where indicated with cAMP. Samples were centrifuged to
equilibrium in a Beckman XL-A analytical ultracentrifuge equipped with
an AN-60 rotor. Absorbance values were measured at 280 nm as functions
of radial position. Five scans were averaged for each sample at each
rotor speed. The approach to equilibrium was considered to be complete
when replicate scans separated by 6 h were
indistinguishable. Solvent densities were measured using a Mettler
density meter.
At sedimentation equilibrium, the absorbance at a specified wavelength
and position in the solution column is given by Equation 1 (11,
12).
![]() |
(Eq. 1) |
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sedimentation Analysis Shows that CAP and lac Repressor Form a 2:1
Complex in the Absence of cAMP--
Representative sedimentation
profiles of lac repressor and CAP, obtained at 4 °C and
18,000 rpm, are shown in Fig.
2A. The solid curves through
the data are global least-squares fits of the expression for a single
species (Equation 1 with n corresponding to lac
repressor or CAP, as appropriate) to six data sets (2 concentrations, 3 rotor speeds) for each protein, obtained using the program NONLIN (34).
The small, symmetrically distributed residuals demonstrate the
compatibility of the single-species model with the data. The molecular
weights returned by these analyses were 48,580 ± 1,760 for
CAP and 153,498 ± 6,950 for lac repressor. The
agreement with the molecular weights derived from sequence
(Mr (CAP dimer) = 47,238;
Mr (lac repressor tetramer) = 154,520) indicates that neither protein is significantly degraded nor
aggregated, under our experimental conditions.
|
Solutions containing both CAP and lac repressor contained additional species that gave weight-average molecular weights significantly greater than those expected for noninteracting proteins. Shown in Fig. 2B is data acquired with such a mixture. The smooth curve represents the fit of Equation 1 (with terms for free CAP, free repressor, and a CAP-repressor complex) to the data. In this fit, Mr values of CAP and repressor were fixed but that of the third species was allowed to float, returning a value of 240,820 ± 14,140. This is within error equal to the value (248,996) expected for a 2:1 CAP/repressor complex. The small residuals attest to the compatibility of this model with the data, although the upward deviation of residuals at the bottom of the cell suggests that higher molecular weight species can form at very high [protein]. This result is the first demonstration, to our knowledge, of a direct interaction of CAP with lac repressor, in the absence of DNA.
Other models for the complex were tested, including a 1:1 CAP/repressor
complex, a 3:1 CAP/repressor complex, self-association of CAP (with no
repressor binding), and self-association of lac repressor
(with no CAP binding). All of these models fit the data significantly
less well than the 2:1 CAP/repressor model, and all gave larger
residuals with nonrandom distributions (results not shown). In
addition, a model with terms for free CAP, free lac
repressor, a 1:1 complex, and a 2:1 complex fit the data as well as the
3 species model, but returned values of the concentration term for the
1:1 complex (1:1,0) that were within error equal to
zero. Whereas this does not rule out the presence of a 1:1 complex in
these reaction mixtures, it indicates that it does not accumulate to
significant levels.
The continuous variation (Job) method (13) was used to further
test the 2:1 complex model (Fig. 2C). Eight samples prepared with total protein fixed ([CAP] + [lac repressor] = 3.4 × 106 M), but ranging in
mole-fraction of CAP from 0.2 to 0.9, gave values of
Mr (complex) compatible with a 2:1 molar ratio
(mean ± S.D. = 243,600 ± 8,150; Fig. 2C,
upper panel). In addition, the amount of complex observed
depended on the mole-fraction of CAP, giving a maximum near X(CAP) = 0.7 (equivalent to a molar ratio of 2.3 CAP/repressor). These results
confirm that the dominant complex has a 2:1 stoichiometry, and the
preferential formation of 2:1 complex and the absence of detectable 1:1
complex at low CAP/repressor ratios strongly suggests that the binding
of CAP to lac repressor is cooperative.
Complex Formation Is cAMP-dependent-- A general mechanism for CAP-repressor binding in the presence of cAMP is shown in Reaction 1,
![]() |
![]() |
![]() |
(Eq. 2) |
|
CAP Does Not Bind Dimeric lac Repressor--
A parallel
analysis was carried out with the lacI-18 mutant lac
repressor. This protein lacks 18 C-terminal residues that mediate
tetramer formation in the wild-type protein (17, 18). The result is a
dimeric repressor protein that binds one equivalent of DNA but cannot
bridge between two DNA segments in the fashion observed with wild-type
repressor (19-22). Shown in Fig. 4 are sedimentation data for the lacI-18 repressor alone and for a
representative sample containing lacI-18 repressor and CAP. The data
for repressor alone are well fit by a single-species model, with a
weight-average molecular weight of 74,280 ± 3,780, in close
agreement with that predicted for the dimer (Mr = 77,260). However, inclusion of CAP in the solution does not result in
a detectable concentration of complex. The data are consistent with a
two-species model, which returns Mr values of
50,030 ± 3,740 for CAP and 76,140 ± 4,350 for repressor, in
good agreement with their individual molecular weights. Models with
terms for CAP, lacI-18 repressor, and a CAP-repressor complex fit the
data as well as the two-species model, but returned values of the
concentration term for the complex (CR,0) that were
indistinguishable from zero. Although these results do not rule out
complex formation, they indicate that none accumulated to a level
sufficient for detection.
|
A Model of the CAP2·lac Repressor
Complex--
Because our preparations of lacI-18 repressor appeared
homogeneous and of correct molecular weight by SDS-PAGE and
sedimentation equilibrium criteria and were active in DNA binding (5,
8), it seems unlikely that their inability to bind CAP is a consequence of either proteolytic degradation or gross misfolding of the protein. We therefore hypothesize that the difference in CAP-binding activity of
dimeric and tetrameric repressors is a functional difference of the
native proteins. The repressor tetramer structure (Fig. 5) reveals two features not present in
the dimer that we propose as candidates for CAP-interaction sites.
These are the C-terminal structures that organize repressor as a dimer
of dimers (and which are disrupted by the lacI-18 mutation), and the
cleft between adjacent dimeric repressor assemblies. The approximate
2-fold symmetry of the tetramer implies that two copies of these
structures are available to bind two equivalents of CAP, as was
observed. Also shown in Fig. 5 is a schematic model of a CAP-repressor
complex in which two molecules of CAP are depicted interacting with the cleft between adjacent dimeric repressor assemblies.
|
Current models of lac promoter regulation (see Refs. 23-26) focus on the independent functions of CAP and lac repressor at their primary binding sites (CAP site 1 and operator 1). These models reflect the absence of clear evidence for regulatory cross-talk between CAP and repressor in the extensive literature dedicated to this system. One might well ask why such cross-talk is not seen, given the overlap of binding sites in the lac promoter region (Fig. 1), and the in vitro data indicating that CAP interacts with lac repressor (described above). The results presented in this paper may be a step toward the answer to that question. As a working hypothesis, we propose that formation of energetically favorable CAP-repressor contacts may compensate for the loss of preferred CAP-DNA contacts when CAP is displaced from operator 1 by lac repressor. These interactions could reduce the CAP potential to interfere with the regulatory repressor-operator 1 interaction. By the same token, favorable CAP-repressor interaction may reduce the overall energetic cost of the isomerization of the repressor-operator 3 complex that occurs when CAP binds CAP site 1 (6). These interactions could reduce repressor's potential to interfere with the regulatory interaction of CAP with CAP site 1. These considerations predict new classes of CAP and lac repressor mutations that should allow testing of this hypothesis in vivo. Such mutations would disrupt the protein-protein interaction but not the corresponding DNA binding activities. Analogous mutations affecting CAP-RNA polymerase- and CAP-CytR repressor-interactions have been identified (25, 27). If this hypothesis is correct, destabilization of the CAP-repressor complex should result in lac promoter repression that depends on the absence of cAMP and CAP, and transcription activation by CAP + cAMP that depends on the absence of lac repressor.
Finally, this is the first demonstration, to our knowledge, of a direct
interaction of CAP with lac repressor, in the absence of
DNA. Little is known about the location or functions of these proteins
when they are not bound to their regulatory DNA sequences. The results
presented here raise the possibility that CAP and lac
repressor may be associated when not performing their
transcription-regulatory functions.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Kathleen S. Matthews for providing the wild-type and mutant lac repressor proteins used in this study.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 717-531-5250;
Fax: 717-531-7072; E-mail: mfried@psu.edu.
§ Present address: Dept. of Biochemistry, Given B409, The University of Vermont College of Medicine, Burlington, VT 05405.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M009087200
2 M. Fried, unpublished results.
3 For the reaction shown in Equation 2, net cAMP uptake will yield n > 0, net release, n < 0, and a cAMP-independent reaction, n = 0.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CAP, cyclic AMP receptor protein; PAGE, polyacrylamide gel electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Straney, S. B., and Crothers, D. M. (1987) Cell 51, 699-707[Medline] [Order article via Infotrieve] |
2. | Lee, J., and Goldfarb, A. (1991) Cell 66, 793-798[Medline] [Order article via Infotrieve] |
3. | Schlax, P. J., Capp, M. W., and Record, M. T., Jr. (1995) J. Mol. Biol. 245, 331-350[CrossRef][Medline] [Order article via Infotrieve] |
4. | Hudson, J. M., and Fried, M. G. (1990) J. Mol. Biol. 214, 381-396[Medline] [Order article via Infotrieve] |
5. | Vossen, K. M., Stickle, D. F., and Fried, M. G. (1996) J. Mol. Biol. 255, 44-54[CrossRef][Medline] [Order article via Infotrieve] |
6. | Fried, M. G., and Hudson, J. M. (1996) Science 274, 1930-1931[Medline] [Order article via Infotrieve] |
7. | Fried, M. G., and Crothers, D. M. (1984) J. Mol. Biol. 172, 241-262[Medline] [Order article via Infotrieve] |
8. | Stickle, D. F., Liu, G., and Fried, M. G. (1994) Eur. J. Biochem. 226, 869-876[Abstract] |
9. |
Anderson, W. B.,
Schneider, A. B.,
Emmer, M.,
Perlman, R. L.,
and Pastan, I.
(1971)
J. Biol. Chem.
246,
5929-5937 |
10. | Butler, A. P., Revzin, A., and von Hippel, P. H. (1977) Biochemistry 16, 4757-4765[Medline] [Order article via Infotrieve] |
11. | McRorie, D. K., and Voelker, P. J. (1993) Self-Associating Systems in the Analytical Ultracentrifuge , Beckman Instruments, Inc., Palo Alto, CA |
12. | Laue, T. M., and Stafford, W. F., III. (1999) Annu. Rev. Biophys. Biomol. Struct. 28, 75-100[CrossRef][Medline] [Order article via Infotrieve] |
13. | Huang, C. Y. (1982) Methods Enzymol. 87, 509-525[Medline] [Order article via Infotrieve] |
14. | Wyman, J., and Gill, S. J. (1990) Binding and Linkage , pp. 63-121, University Science Books, Mill Valley, CA |
15. | Takahashi, T., Blazy, B., and Baudras, A. (1980) Biochemistry 19, 5124-5130[Medline] [Order article via Infotrieve] |
16. | Kolb, A., Busby, S., Buc, H., Garges, S., and Adhya, S. (1993) Annu. Rev. Biochem. 62, 749-796[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Chen, J.,
and Matthews, K. S.
(1992)
J. Biol. Chem.
267,
13843-13850 |
18. | Chen, J., Surendran, R., Lee, J. C., and Matthews, K. S. (1994) Biochemistry 33, 1234-1241[Medline] [Order article via Infotrieve] |
19. |
O'Gorman, R. B.,
Dunaway, M.,
and Matthews, K. S.
(1980)
J. Biol. Chem.
255,
10100-10106 |
20. | Krämer, H., Niemöller, M., Amouyal, M., Revet, B., von Wilcken-Bergmann, B., and Müller-Hill, B. (1987) EMBO J. 6, 1481-1491[Abstract] |
21. | Chakerian, A. E., and Matthews, K. S. (1992) Mol. Microbiol. 6, 963-968[Medline] [Order article via Infotrieve] |
22. | Oehler, S., Amouyal, M., Kolkhof, P., von Wilcken-Bergmann, B., and Muller-Hill, B. (1994) EMBO J. 13, 3348-3355[Abstract] |
23. | Reznikoff, W. S. (1992) Mol. Microbiol. 6, 2419-2422[Medline] [Order article via Infotrieve] |
24. | Reznikoff, W. S. (1992) J. Bacteriol. 174, 655-658[Medline] [Order article via Infotrieve] |
25. | Ebright, R. H. (1993) Mol. Microbiol. 8, 797-802[Medline] [Order article via Infotrieve] |
26. | Muller-Hill, B. (1996) The lac operon , Walter de Gruyter, Berlin |
27. | Sogaard-Andersen, L., Mironov, A. S., Pedersen, H., Sukhodelets, V. V., and Valentin-Hansen, P. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4921-4925[Abstract] |
28. | Majors, J. E. (1977) Control of the E. Coli Lac Operon at the Molecular Level. Dissertation , Harvard University |
29. | Galas, D. J., and Schmitz, A. (1978) Nucleic Acids Res. 5, 3157-3170[Abstract] |
30. | Schmitz, A. (1981) Nucleic Acids Res. 9, 277-292[Abstract] |
31. | Malan, T. P., and McClure, W. R. (1984) Cell 39, 173-180[Medline] [Order article via Infotrieve] |
32. | Lewis, M., Chang, G., Horton, N. C., Kercher, M. A., Pace, H. C., Schumacher, M. Z., Brennan, R. G., and Lu, P. (1996) Science 271, 1247-1254[Abstract] |
33. |
Berman, H. M.,
Westbrook, J.,
Feng, Z.,
Gilliland, G.,
Bhat, T. N.,
Weissig, H.,
Shindyalov, I. N.,
and Bourne, P. E.
(2000)
Nucleic Acids Res.
28,
235-242 |
34. | Johnson, M., Correia, J. J., Yphantis, D. A., and Halvorson, H. (1981) Biophys. J. 36, 575-588[Abstract] |