The gal operon of bacteria encodes enzymes of galactose metabolism that constitute an

amphibolic pathway and is transcribed by two promoters. Several cis-acting DNA sequences
(control elements) and a variety of regulatory proteins, including a histone-like protein, modulate
the two promoters in a multitude of ways usually found in animals and not in bacteria (Table 1).
The gal operon has revealed several new features of gene-specific transcriptional regulation that
were previously unrecognized. Perhaps such multivalent control mechanisms are required to
regulate the synthesis of amphibolic enzymes.
Table 1. Repression and Activation of Transcription of the gal Operon
Promoters Regulatory protein and DNA elements
GalR, HU GalR or cAMPCRP
GalS
AS
O O
% I E

PI Repression Repression Activation

P2 Repression Activation Repression
a
The regulatory effects of GalR and GalS are epistatic to the effects of cAMP*CRP. 1. Enzymes
of the gal Operon
The galactose metabolizing enzymes (Fig. 1) are involved in (i) the catabolism of D-galactose
that was either imported into the cell by permeases or generated intracellularly by hydrolysis of
disaccharides and (ii) the synthesis of precursors (UDP galactose and UDP glucose) of complex
carbohydrates (1, 2). For catabolism, only a-D-Galactose is converted to galactose-1-phosphate
by galactokinase (3). b-D-galactose, generated, for example, by hydrolysis of lactose by beta-
galactosidase, must change to the a-anomer before it can be phosphorylated. Although b-D-
galactose can mutarotate spontaneously to the a-anomer at a slow rate, the enzyme aldose-1-
epimerase is largely responsible for the mutarotation in vivo (2). Thus, aldose-1-epimerase links
the enzymes of lactose and galactose metabolism into a common pathway (Fig. 1).
Figure 1. The Leloir pathway of D-galactose metabolism. As shown, D-galactose is
generated intracellularly by hydrolysis of the disaccharide lactose. The parts of the
pathway catalyzed by enzymes of the gal operon are shown in bold. They are encoded by
the genes shown within the parenthesis in italics.
2. Regulation of Transcription
The structural genes and the associated regulatory elements of the gal operon are shown in
Figure 2. The gal operon is transcribed from two promoters, P1 and P2, which are separated by
5 base pairs (bp) (4, 5). The dominant regulator of the gal operon is the Gal repressor (GalR) (6).
GalR, together with the histone-like protein, HU, acting as a corepressor, keeps the expression
from the gal promoters low (7, 8). The operon is induced 15-fold in the presence of D-galactose
or some of its nonmetabolizable analogs, such as D-fucose (9). The inducer lifts the repression
by an allosteric effect on GalR. It is not known whether the true inducer is the a- or the b-anomer
of the sugar, or both. Besides the major negative control by GalR and HU, the two promoters are
also regulated by GalR alone (10), by a Gal isorepressor, GalS (11-13), and by a complex of
cyclic AMP (cAMP) bound to cyclic AMP receptor protein (CRP), cAMP*CRP (4, 5, 14-16). As
described below, P1 and P2 are regulated by these regulators in opposite directions. The gal
promoters are also modulated by cis-acting DNA sequences without the participation of any
regulatory proteins.
Figure 2. The structure of the gal operon. The regulators are shown as circles and their
cognate DNA control elements as open bars. Their mode of actions are explained in the
text. The adenine tracks present upstream of the promoter are not shown.

3. Regulation without Regulatory Proteins

3.1. Control of P1 by Adenine Tracks

The intrinsic strengths of the two promoters are comparable, and they are moderately active in
the absence of any regulatory proteins, both in vivo and in vitro. In vitro, the intrinsic strength of
P1 is twofold enhanced because of the periodic presence of four to six adenine residues
centered at positions -84.5,-74and-63 on the DNA (17). The adenine tracks bend the DNA toward
the face of P1 to which RNA polymerase binds. The DNA curvature induced by the adenine
tracks may help formation of a RNA polymerase-promoter complex (caging) that is more optimal
for transcription initiation at P1 (18).

3.2. Control of P2 by UTP

Transcription of the gal operon from the P2 promoter is very low when the concentration of UTP
is high and vice versa, both in vivo and in vitro (19). UTP regulates the step of promoter
clearance by RNA polymerase at the P2 promoter in an intriguing way. In vitro, RNA polymerase
clears at P2 poorly and makes a large amount of aborted RNA oligomers, unlike at P1. At high
concentrations of UTP, it also synthesizes pseudo-templated RNA oligomers of the composition

because the enzyme "stutters" while adding uridine residues present at positions 3-5 of the P2
RNA and not the P1 RNA. RNA polymerase clears the promoter more efficiently and makes
template-encoded normal gal RNA at low UTP concentrations. The involvement of UTP in the
synthesis of UDP sugars by the galactose pathway may be the reason for the control by UTP
(Fig. 1) analogous to the way UTP controls its own synthesis in the pyrimidine operons (20). At
high UTP concentrations, the levels of UDP galactose and UDP glucose are high and inhibit the
synthesis of enzymes, specifically, galactose-1-phosphate uridyltransferase and uridine
diphosphogalactose epimerase, that make them.

4. Repression by GalR and HU: DNA Looping

Transcription from the two gal promoters is coordinately repressed (negative control) by GalR
and HU acting together (7, 8). The repression requires binding of GalR to two operators, Oe and
O which are similar 16-bp sequences with a dyad symmetry (Fig. 2) (21-23). O e is located
upstream of the promoters at position _60.5, whereas O i is located within the structural gene
galE at position +53.5. The two operator-bound GalR molecules associate with each other in the
presence of the cofactor, HU (a heterodimer of two subunits, HUa and HUb), resulting in the
formation of a DNA loop encompassing the promoters (8, 24) (Fig. 3). The DNA looping
presumably changes the structure of the promoters, making them refractory to RNA polymerase
caging (18, 25). The nucleoprotein complex that represses the gal promoters is called the Gal
repressosome, because its formation is by binding of a histone-like component of the bacterial
nucleoid as well as a repressor (26). Although GalR binds to Oe and Oi in both the absence and
presence of HU, GalR binding alone does not bring about DNA looping and the associated
simultaneous repression of the promoters (27). HU is absolutely necessary for the effect; it
cannot be replaced by other histone-like proteins. Although HU is not a sequence-specific DNA
binding protein, one molecule of HU heterodimer binds to and bends the gal DNA centered at an
architecturally critical position (8). Genetic analysis and modeling defined the GalR surfaces
interacting to form a stacked, V-shaped tetrameric structure (28, 29). Evaluation of the DNA
elastic energies gave unambiguous preference to a DNA loop in which OE and OI adopt an
antiparallel orientation causing undertwisting of DNA (29). Since HU binding depends on GalR
binding to both Oe and Oi and the binding of GalR to the operators is enhanced by HU, GalR and
HU bind cooperatively (8). GalR piggybacks HU to the critical position on the DNA through a
specific GalR-HU interaction (31). The entire process facilitates the GalR-GalR interaction
resulting in cooperativity. The GalR-HU contact may be transient and was not in the final
repressosome structure. The dependence of HU binding on GalR renders the HU-containing
nucleoprotein complex that brings about repression of P1 and P2 sensitive to inducer D-
galactose for transcription derepression. Such a mechanism is an example of how DNA that is
"condensed" by binding proteins and made refractory to RNA polymerase action can become
available for transcription in response to specific signals.
Figure 3. The DNA looping of the gal promoter by binding of the GalR regulator and HU
corepressor. The details of binding of the two proteins are discussed in the text.

5. Regulation in the Absence of DNA Looping: Interaction between GalR

and RNA Polymerase
Without DNA looping (ie, in the absence of HU), occupation of Oe alone by GalR represses P1
(by about four- to fivefold) and activates P2 (twofold) (10, 27). Binding to O i does not affect this
dual control. The activation of P2 and repression of P1 are independent of each other. GalR
exerts its specific regulatory effect on one promoter even when the other is mutated. The
activation of P2 or repression of P1 is not an intrinsic property of the promoter; the regulation can
be reversed by switching the angular orientation of the promoters relative to O e by inserting a 5-
bp segment, that is, half of a DNA helix, between OE and the promoters. Both activation of P2
and repression of P1 require the formation of a specific GalR-RNA polymerase-DNA complex at
each promoter. RNA polymerases containing a subunits that carry specific amino acid alterations
in their carboxy terminal domain (aCTD), or that are missing the aCTD, abolish the regulatory
effect of GalR without affecting intrinsic transcription, suggesting that GalR activates and
represses by a direct contact with the promoter-bound RNA polymerases.

5.1. Activation of P2
Activation of P2 by GalR bound to DNA at position _60.5 (_55.5 with respect to P2) parallels the
activation of transcription of several promoters by activators that also bind around position _60
on the DNA and act by enhancing open complex formation (32). An exposed segment (not
necessarily the same segment) of aCTD is contacted by the DNA-bound activator to activate
transcription (33, 34). aCTD is connected to the rest of the RNA polymerase molecule by a
flexible hinge (35, 36). In P2, as in these systems, aCTD binds at the region 40 bp upstream of
P2 (37) (Fig. 4).

Figure 4. Repression of P1 promoter and activation of P2 promoter by a contact between

DNA-bound GalR and the aCTD of RNA polymerase. The two domains aNTD and aCTD of
the RNA polymerase a subunit are shown shaded. The aCTD is connected to the rest of
RNA polymerase by a flexible hinge. Note the differences in the topography of the two
cases.
5.2. Repression of P1
GalR occupying DNA at position -60.5 represses P1, not by hindering RNA polymerase binding,
but by contacting aCTD, which binds to P1 at position 45 bp upstream of P1 (10, 27, 37) (Fig. 4).
GalR inhibits isomerization of RNA polymerase complex at P1. How contacts between the same
two proteins bring about opposite effects at the two promoters remains to be determined. It is not
known why and under what conditions the dual behavior of GalR toward P1 and P2 in the
absence of DNA looping is triggered in cells.

6. Gal Isorepressor
The gal operon is also regulated by an isorepressor (GalS). Although GalS does not seem to
repress the gal promoters by DNA looping, the isorepressor does stimulate P2 and repress P1,
the same way that GalR does by binding to OE, except that the effects are weaker. GalR and
GalS modulate a few other operons, including those encoding both high- and low-affinity
galactose active transport systems (12, 13, 38). The degree of regulation by GalR and GalS
varies from operon to operon, perhaps to coordinate galactose metabolism and to transport
efficiently under a wide range of galactose availability.

7. Properties of GalR and GalS

GalR and GalS are 85% similar in their amino acid sequences (12). By their homology with
proteins (GalR-LacI family) whose structures are known, GalR and GalS are expected to have
two-domains connected by a flexible hinge (39). A helix-turn-helix motif in the amino domain of
each subunit of GalR and GalS dimers recognizes half of a dyad symmetry in OE and OI. The
carboxy domains contain the inducer binding sites, which have been defined by isolation and
characterization of inducer nonbinding (noninducible) repressor mutants, such as galRs and by
modeling (40, 41). The mechanism by which the inducer derepresses the P1 promoter under
conditions of nonlooping by binding to the ^-bound GalR has been studied in detail. Despite the
demonstration that inducer binding can dissociate GalR from the operator, P1 can be
derepressed under conditions when O E is occupied by a GalR-inducer complex (42). These
results show that dissociation of a repressor from an operator is not obligatory for transcription.
Since the OE-bound GalR interacts with RNA polymerase to inhibit open complex formation at
P1 at a postbinding step, the inducer acts by allosterically neutralizing the inhibitory contact
between the proteins without dissociating the repressor from DNA (10, 17).

8. Regulation by cAMP*CRP
Like GalR and GalS, the global regulator cAMP*CRP complex also has differential effects on the
gal promoters; but unlike GalR and GalS, the complex activates transcription from P1 (three- to
fourfold) and represses the same from P2 (10-fold) (14, 16). Unlike a large group of promoters,
such as the lac promoter, in which cAMP*CRP dimer activates transcription by binding at position
_61.5 on DNA and contacting RNA polymerase through the aCTD (see Lac Operon and Cyclic
Amp Receptor Protein (CRP)/Catabolite Gene Activator Protein (CAP)), the regulatory complex
brings about dual control in the gal operon by binding to DNA at position _41.5 (37, 43, 44) (Fig.
5). Whereas it is believed that cAMP*CRP represses P2 by sterically hindering RNA polymerase
binding to the overlapping _35 element of the promoter, the molecular mechanism of activation of
P1 is different from that in the lac promoter. cAMP*CRP stimulates transcription initiation at P1 by
stimulating both RNA polymerase binding (closed-complex formation) and isomerization (45). A
patch of amino acid residues (called region 1) of the promoter distal subunit of the cAMP*CRP
dimer interacts with an a-helix (helix 1) of the aCTD and helps the latter to stretch away from the
N-terminal domain (aNTD) to bind to the region upstream of cAMP*CRP. A different amino acid
residue patch (called region 2) in the promoter proximal subunit of cAMP*CRP interacts with a
different segment of the aNTD. The contact with the aCTD is responsible for increasing the RNA
polymerase binding, whereas the interaction with the aNTD stimulates the isomerization step.
This shows how the same regulatory protein can activate transcription initiation at two different
biochemical steps by making entirely different contacts with RNA polymerase. Nevertheless, the
dual roles of cAMP*CRP enable the gal operon to be expressed primarily from P1 in cAMP-
proficient cells and from P2 in cAMP-deficient (eg, glucose grown) cells.
Figure 5. Activation of P1 by cAMPCRP by two different contacts with RNA polymerase.
The two domains aNTD and aCTD of the RNA polymerase a subunit are shown shaded.
Details are discussed in the text.
9. Summary
The study of the gal operon in E. coli has shown new ways and means of gene regulation at
the level of transcription initiation: (i) how proteins bound to spatially separated sites on DNA
communicate by DNA looping; (ii) how specific and nonspecific DNA binding proteins cooperate
to "condense" DNA, making the latter unavailable for transcription, while remaining sensitive to
an inducing signal; (iii) how the same regulator brings about opposite effects on promoters
activation and repressionby making direct contact with RNA polymerase; and (iv) how an
inducer allosterically changes a repressor to neutralize the inhibitory effect of repressor still
bound to DNA. Besides studying the detailed biochemical mechanisms of such controls, the two
interesting questions remain: What are the physiological reasons for the multitude of controls,
and how are these diverse controls coordinated in the cell?

N E X T P O S T: Gamma-Aminobutyric Acid (GABA) (Molecular Biology)

P R E V I O U S P O S T: G-Protein-Coupled Receptors (Molecular Biology)