From the Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255
Activation and repression of transcription are
primarily caused by gene regulatory proteins (activators and
repressors), which act by binding to specific sites on DNA. The steps
from initial binding of RNA polymerase to the elongating complex are
characterized by many intermediates, each with a discrete structure,
offering many mechanistic possibilities for regulator actions. It has
been shown in some systems that the activator acts by helping RNA
polymerase or other associated factors to bind (recruitment) and/or by
influencing a postrecruitment step (isomerization, promoter clearance,
etc.) (1-7). We have used the term recruitment for referring to
assistance only on the initial binding step of RNA polymerase. We
caution that a postbinding step may be indistinguishable from the
recruitment step if they are in rapid equilibrium. Clearly, all
activators do not act at the level of RNA polymerase recruitment to the
promoters. There are activators demonstrated to help postbinding steps
that have no effect on initial binding (4-7). Promoter-specific
repression can occur by sterically hindering the binding of RNA
polymerase or of, in principle, another essential transcription factor
to the promoter (8, 9). However, other studies in several promoters, as
was anticipated (10), point toward repressor action also through
contact with promoter-bound RNA polymerase at a postbinding step
(11-17). More interestingly, some regulators act as activator in one
context and as repressor in another (13, 15). Although the contact
regions on the surface of some regulators and of RNA polymerase have
been mapped (18, 19), how these contacts cause activation or inhibition
of transcription initiation in biochemical terms is not known. In
principle, the contact may affect the process of transcription
initiation (i) by allosteric modification of RNA polymerase and/or (ii)
by energetic stabilization of an intermediate(s). Regulator-induced
conformation changes in RNA polymerase by protein-protein contact may
contribute to the regulation process. However, a regulator-RNA polymerase contact may play a fundamentally different role in transcription initiation. In this article, we provide a conceptual framework for the process of activator and repressor action through differential stabilization of one or more of the intermediate states of
RNA polymerase-promoter complex by its contact with the regulator. We
portray regulators as catalysts. From a thermodynamic point, we view
that activators, like catalysts, lower the activation energy of some
step(s) in the reaction pathway of transcription initiation. As
discussed below, a similar energetic argument explains the action of
repressors. To make our point, we discuss simple examples of
DNA-binding regulators modulating RNA polymerase during transcription
initiation in selected prokaryotic systems.
The biochemical steps of RNA polymerase binding to the promoter
leading to transcription initiation have been discussed extensively (20-22). In principle, any of the steps can be regulated; a
rate-limiting step can easily be enhanced by an activator or quenched
by a repressor (10, 23, 24). To describe the role of a regulator in the simplest way, we will use, for example, the open complex formation as a
two-step chemical reaction that includes the formation of only one
transition state,
INTRODUCTION
Top
Introduction
References
Regulators as Catalysts
where R is RNA polymerase, P is promoter DNA, (R·P)c is
the closed complex of RNA polymerase and DNA; (R·P)o is the
open complex of RNA polymerase and DNA; [R·P]
is the transition state between (R·P)c and (R·P)o,
Ka is the equilibrium constant that characterizes
the closed complex, and kf is the rate constant of
the isomerization from closed to open complex. Thus, the reactions can
be described by a free energy diagram (25), which has been very useful
in explaining catalysis by enzymes that act by lowering the energy
barrier(s) during the course of a reaction (26-29). We propose that
both activator and repressor modulate the energetics of the reactions
steps. This outlook of a regulator action not only provides a common biochemical and thermodynamic basis of its action but also addresses the following questions. (i) What role does DNA play in regulator action? (ii) How can a regulator be bifunctional, i.e.
activator in one context and repressor in another?
Fig. 1 represents a minimal kinetic scheme of a typical open complex formation reaction at a hypothetical promoter that is not regulated, and the associated free energy changes during the course (change in reaction coordinates). RNA polymerase conformation constantly changes depending on the DNA sequence during the course of the reaction (30, 31). We propose a differential contact1 model to explain regulator action. In this model, a given regulator after binding to DNA interacts with and lowers the free energy of one or more of the DNA-bound RNA polymerase intermediates (including the transition state), each with discrete conformation, during the course of open complex formation. The differential protein-protein contacts may persist throughout the progression from closed to open complex and lower the free energy level of all states with respect to unbound RNA polymerase. On the other hand, the contact may be specific for some state(s), selectively stabilizing that intermediate and lowering its free energy. Changes in RNA polymerase conformation during the initiation steps may facilitate differential contacts.
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As explained below, this provides the regulator the option to enhance or inhibit open complex formation by decreasing or increasing the energetic barrier of the different steps. The presence of two proteins on adjacent DNA sites having the potential for interaction does not ensure that they will interact because of the importance of establishing proper geometry. The net interaction energy and consequent stabilization is a function of several factors: local concentration of the regulator (entropic assistance), free energy of regulator-RNA polymerase interaction, and any required protein and DNA distortion energy. If the geometry is highly unfavorable, the required DNA and protein distortion energy will not be compensated by the entropic gain and protein-protein interaction energy for proper regulator-RNA polymerase contact or stabilization. The variation of orientations of two adjacently bound proteins during the progress of the reaction can permit contacts in some orientation and not in others (differential contacts).
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Activation |
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In the example of an activator action, the concentration of
unbound RNA polymerase and free DNA, whose free energy is taken as the
reference state, is assumed to be slightly higher than the true
Ka1 for closed complex formation, and
the closed complex is placed at a slightly lower free energy level than
that of the unbound RNA polymerase and free DNA (Fig.
2, A-C, black
lines). For simplicity, we have not included a transition
state intermediate for closed complex formation and made the transition
state of isomerization the rate-limiting step by placing [R·P]
at the highest free energy level. A regulator can activate
transcription by any of the scenarios described in the following
paragraphs.
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Plot 1--
The regulator has contacts with (R·P)c and
[R·P] with the same affinity and lowers their free energy by
equal amounts during the progress of the reaction (Fig. 2A,
green line). As shown in the free energy diagram,
such interactions will decrease only
Gc, and not
G
, the net result being activation because of
increased Ka. The net rate enhancement of open
complex formation will be most significant if the free energy of
(R·P)c in the absence of activator is higher than that of
unbound RNA polymerase, i.e.
Ka
1 is greater than the unbound RNA
polymerase concentration. If the RNA polymerase concentration is higher
than Ka
1, then further stabilization
of the closed complex by contact with a regulator would not
significantly enhance the net rate of open complex formation. Thus, the
maximal effect of such a regulator can only be observed if the RNA
polymerase concentration is below Ka
1.
A contact between the C-terminal domain of the
-subunit
(
CTD)2 of RNA polymerase
and the downstream subunit of CRP was shown in a study employing wild
type and mutant proteins where a correlation was established between
the ability of CRP to interact with RNA polymerase in solution and its
ability to activate the lac promoter (32). It has been shown
that CRP activates lac transcription by increasing the
pseudoequilibrium constant3
as derived from kinetic measurements (2), implying that the contact
between CRP and
CTD is held at both closed and transition state
complexes with approximately equal free energy.
Plot 2--
The regulator interacts and lowers the free energy of
both (R·P)c and [R·P] but decreases the free energy of
[R·P]
more than that of (R·P)c (Fig. 2B,
green line). The result will be decreases in both
Gc and
G
and thus activation by
increasing both Ka and kf. This
type of regulation is exemplified by
cII at the
PRE and PINT promoters
(33, 34) and by CRP activation of the gal P1 promoter (35,
36). If, on the other hand, the regulator decreases
Gc more than it decreases
G
, the
situation will be as described below under "Repression" (Plot
4).
Plot 3--
The regulator makes differential contacts and lowers
the free energy of [R·P], thus decreasing
G
.
The net effect is activation because of an increase in
kf (Fig. 2C, green
line). The stronger the interaction, the lower will be the
activation energy for the formation of the transition state and the
higher will be kf. RNA polymerase concentration
would not have any effect on this type of activator action.
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Repression |
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The general nature of repression by contact with RNA polymerase is likely to be stabilization, by differential contact, of any of the intermediates with respect to the subsequent transition state. The bound RNA polymerase faces an increased energetic barrier to the next step as exemplified by Plot 4.
Plot 4--
In the free energy diagram in Fig.
3, a promoter is described that proceeds
without much of an energy barrier. The regulator interacts and lowers
the free energy of (R·P)c only and not of [R·P], thus
increasing
Gc as well as
G
.
Although there will be an accumulation of closed complex
(R·P)c (increase of
Gc),
i.e. an increase of Ka, a large increase
in
G
will create an energy trap for the isomerization step. If RNA polymerase concentration is greater than
Ka
1, the corresponding decrease in
kf overcompensates for the increase in
Ka, and the net effect will be repression. GalR, the
repressor of the Escherichia coli gal operon, clearly does
not act by competing with RNA polymerase for DNA binding (13, 43, 44)
as has been suggested for LacI (9). GalR and RNA polymerase form a
stable ternary complex at the galP1 promoter, and repression
of P1 is abolished either by truncation of or by specific
amino acid alterations of the
CTD of RNA polymerase. These results
show that GalR inhibits RNA polymerase activity at a postbinding step
through a direct contact with
CTD. This phenomenon is easily
explained if GalR makes a differential contact with the closed complex
in the free energy diagram (Fig. 3, red line), a
net decrease in kf.
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The Role of DNA |
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What part does DNA play in the action of a regulator? Several
roles have been suggested before (25, 45). (i) Having a regulator-specific DNA binding site brings specificity to the regulator. Most DNA-binding gene regulatory proteins have unique sequences to which they bind. (ii) DNA binding near a promoter increases the local concentration of the regulator. (iii) DNA binding
changes the structure of the regulator allosterically making it more
"potent" for differential contacts. (iv) DNA bending induced by a
regulator can directly affect transcription initiation by correctly
aligning interacting groups of proteins and DNA in space. Although some
or all of these roles of DNA may be essential for the function of a
given regulator, the following suggests that such roles may not be
sufficient (46). When a 4-nucleotide single-stranded gap is introduced
in the DNA segment intervening the CRP binding site and the
lac promoter, CRP not only failed to activate but also
repressed the basal transcription. DNA binding results, nevertheless,
showed normal, if not better, formation of CRP·DNA·RNA polymerase
ternary complex under such conditions, demonstrating that
protein-protein contact-mediated RNA polymerase recruitment was not
affected by the DNA structural alteration. It was proposed that either
the CRP·RNA polymerase complex is formed by a wrong, i.e.
nonproductive, contact or the intervening DNA plays a more direct role
and must be normal and intact. The differential contact model suggests
that both proposals are valid. In this model, double-stranded DNA
provides the proper rigidity to the nucleoprotein complex, allowing
only the desired contacts. On the other hand, the single-stranded DNA
either allows an unwanted contact creating an energy trap because of
its flexibility or prevents an essential contact between the regulator
and RNA polymerase because of change in geometry. Because CRP activates
the lac promoter by increasing Ka (2), as
discussed above, in the differential contact model the CTD contact
by the regulator must be held at both closed and transition state
complexes. By introduction of a single-stranded gap, the intervening
DNA loses rigidity and consequently its ability to make the "right"
contacts shown in Fig. 2A. In this model, CRP now contacts
either (R·P)c more strongly than [R·P]
or contacts
(R·P)c only (Plot 4), leading to repression. Such
stabilization at the (R·P)c state is likely to be
considerable because the establishment of the contact is no longer
required to overcome the DNA distortion energy (47, 48).
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The Dual Behavior of Regulators |
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GalR-- Under specific conditions, binding of the GalR protein to the site OE in the gal operon of E. coli regulates transcription from two promoters P1 and P2 (13, 43, 44). GalR represses transcription from P1, which is located on the same face of DNA as the DNA-bound GalR, and stimulates that from P2, which is located on the opposite side. Gel electrophoretic studies and DNase protection experiments of gal DNA in the presence of GalR and RNA polymerase have shown that GalR forms a characteristic GalR·DNA·RNA polymerase ternary complex at each promoter because of putative interaction between GalR and RNA polymerase.
GalR binding to OE increases open complex formation at P2 by stimulating RNA polymerase binding (13, 43, 44). Although it is not known that GalR stimulation at P2 is through increasing Kb or kf, or both, GalR behavior can be explained by Plots 1, 2, or 3 in Fig. 2. The proposed interaction of RNA polymerase with GalR is throughMerR--
The E. coli transcription regulator, MerR,
provides another example of a context-dependent dual
behavior (17, 51). The differential contacts with RNA polymerase depend
upon the state of the regulator and the DNA sequence. MerR binds to a
site between the 10 and the
35 of the
Pmer TPCAD promoter. In the absence of Hg(II), MerR represses transcription from the promoter. In the
presence of Hg(II), the DNA·MerR RNA polymerase ternary complex is
converted from a state of repression to a state of activation. Thus,
allosterically changing the structure of a regulator can convert it
from a repressor to an activator.
cI--
As previously mentioned,
cI activates the
PRM promoter by enhancing kf,
i.e. by making a contact with [R·P]
. It acts also as
a repressor of the PR promoter. A mutational change in RNA
polymerase can change the effect of cI. Recently, it has been shown
that a mutation in the
subunit of RNA polymerase can cause
cI to
switch from increasing kf to increasing the
pseudo-equilibrium constant Kb (52). Presumably, the
mutation enables cI to interact with RNA polymerase in the (R·P)c in addition to the [R·P]
, leading to a
predominant Kb effect. In this case, the mutation
may change the strength of the contact and/or orientation of the
surface, allowing an interaction at a different point(s) in the
reaction pathway.
29 p4--
Protein p4 of the Bacillus
subtilis bacteriophage
29 repressess the
transcription of early promoters, e.g. A2C, and
simultaneously activates transcription of an A3 promoter for
late genes. Both the activation and the repression require contacts
between p4 and the
subunit of RNA polymerase. Repression of the A2c
promoter is by inhibition of a later step, promoter clearance. The p4
protein allows RNA polymerase to bind but prevents the elongation step of transcription initiation (15). Activation at the A3 promoter is
through stabilization of the closed complex between RNA polymerase and
DNA (12). It has also been shown that p4-mediated regulation also
depends upon the strength of RNA polymerase-promoter interactions; increasing the strength by changing the
35 sequence converts p4 from
an activator to a repressor (53) (see "Plot 4").
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Perspective |
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The basic concept of differential contacts discussed here can easily be applied to any of the steps of not only prokaryotic but eukaryotic complex regulatory systems. Indeed, a number of examples of eukaryotic dual function regulators have been identified (54-58) and are likely to act by differential stabilization of intermediate states through protein-protein contact. The model can be extended to explain the known examples of activator (4) or repressor (15) action at the levels of post-open complex formation of transcription initiation.
The differential contact model of gene transcription can be tested. It predicts that one will be able to isolate mutations in the regulator, RNA polymerase, or promoter, which will define the state(s) of the regulator contacts, and some of the mutations may switch the state to which the regulator binds and alter the regulatory outcome.
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FOOTNOTES |
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* This minireview will be reprinted in the 1997 Minireview Compendium, which will be available in December, 1997.
On leave from the Department of Biophysics, Bose Institute,
Calcutta 700 054, India.
§ To whom correspondence should be addressed. Tel.: 301-496-2495; Fax: 301-480-7687; E-mail: sadhya{at}helix.nih.gov.
1 Differential contacts are transient contacts, which are made and broken in a temporal manner, between DNA-bound regulators and RNA polymerase.
2
The abbreviations used are: CTD, C-terminal
domain of the
-subunit; CRP, cAMP receptor protein.
3
The measured pseudo-equilibrium constant was
k1/(k1 + kf).
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REFERENCES |
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