Galactose (Gal) Operon – Structure, Regulation

What is Gal operon?

• The gal operon is a bacterial operon that encodes galactose-metabolizing enzymes.
• This operon’s gene expression is repressed by the binding of repressor molecules to two operators. These repressors form a DNA loop by dimerizing.
• Loop and exteRNAl operator interference prohibit RNA polymerase from attaching to the promoter, hence preventing transcription.
• In addition, since galactose metabolism in the cell involves both anabolic and catabolic pathways, an unique regulatory system employing two promoters for differential repression has been found and defined inside the gal operon.
• The gal operon is transcribed from two overlapping promoters, P1 and P2, whose transcription start sites are indicated as +1 and 5, respectively.
• As enzymes expressed by the gal operon are required for both catabolic and anabolic metabolisms, each promoter reacts to separate regulators to meet physiological requirements. Both promoters express considerable amounts of intrinsic expression.
• Nevertheless, cAMP and its receptor protein CRP complex (CCC) stimulates P1 but inhibits P2, whereas GalR inhibits P1 and stimulates P2 (details below). CCC binds to a single AS site, whereas GalR binds to two operators.

Structure of Gal operon

• The gal operon of E. coli is composed of four structural genes: galE (epimerase), galT (galactose transferase), galK (galactokinase), and galM (mutarotase), which are transcribed from two overlapping promoters, PG1 (+1) and PG2 (-5), upstream from galE.
• GalE encoding an enzyme that transforms UDP-glucose to UDP-galactose. Even when cells are not utilising galactose as a carbon/energy source, this is necessary for the creation of UDP-galactose for cell wall biosynthesis, namely the cell wall component lipopolysaccharide.
• The GalT gene encodes the galactosyltransferase protein, which catalyses the transfer of a galactose sugar to an acceptor, thereby establishing a glycosidic bond.
• GalK is a gene that encodes a kinase that converts -D-galactose to galactose 1-phosphate.
• galM catalyses the first step of galactose metabolism, the conversion of -D-galactose to -D-galactose.
• The gal operon has two operators: OE (for external) and OI (for internal) (for internal). The former is immediately adjacent to the promoter, and the latter is immediately adjacent to the galE gene (the first gene in the operon).
• These operators bind the outside-the-operator-region-encoded repressor GalR. In order for this repressor protein to operate effectively, the operon also has a histone binding site.
• A second site, known as the activating site, is located downstream of the external operator and upstream of PG2. This site is the binding area for the cAMP-CRP complex, which controls promoter activity and, consequently, gene expression.

The Leloir pathway of D-galactose metabolism

• The Leloir pathway for the metabolism of D-galactose. D-galactose is created intracellularly through the hydrolysis of the disaccharide lactose, as demonstrated.
• The portions of the pathway catalysed by gal operon enzymes are shown in bold. They are encoded by the genes given in italics between parenthesis.
• The galactose metabolising enzymes participate in I the catabolism of D galactose that was either imported into the cell by permeases or created intracellularly by hydrolysis of disaccharides and (ii) the creation of complex carbohydrate precursors (UDP galactose and UDP glucose). galactokinase converts just a-D-galactose to galactose-1-phosphate for catabolism. Before it can be phosphorylated, b-D-galactose, which is produced, for example, by the hydrolysis of lactose by beta-galactosidase, must convert to the a-anomer. Although b-D-galactose can spontaneously mutarotate to the a-anomer at a modest rate, aldose-1-epimerase is primarily responsible for the mutarotation in vivo. Thus, aldose-1-epimerase connects the lactose and galactose metabolic enzymes to a single route.

Regulation of Transcription

• The gal operon is transcribed from two promoters P1 and P2 that are 5 base pairs apart (bp).
• The Gal repressor is the primary regulator of the gal operon (GalR). Together, GalR and the histone-like protein HU, which functions as a corepressor, keep the expression of the gal promoters at a low level.
• In the presence of D-galactose or its nonmetabolizable analogues, such as D-fucose, the operon is stimulated 15-fold.
• Using an allosteric impact on GalR, the inducer alleviates repression. It is unknown if the a- or b-anomer of sugar, or both, is the real inducer.
• The two promoters are also controlled by GalR alone, a Gal isorepressor called GalS, and a compound of cyclic AMP (cAMP) and cyclic AMP receptor protein (CRP), cAMP*CRP.
• As shown below, P1 and P2 are regulated in different directions by these regulators. The gal promoters are also controlled by cis-acting DNA regions independent of regulatory proteins.

Regulation without Regulatory Proteins

Control of P1 by Adenine Tracks

• Both in vivo and in vitro, the two promoters have equal intrinsic strengths and are modestly active in the absence of regulatory proteins.
• Due to the periodic presence of four to six adenine residues centred at positions -84.5, -74, and -63 on the DNA, the intrinsic strength of P1 is increased by twofold in vitro.
• RNA polymerase binds to the face of P1 that is bent by the adenine tracks.
• The DNA curvature generated by adenine tracks may facilitate creation of an RNA polymerase-promoter complex (caging) that is more suited for transcription initiation at P1.

Control of P2 by UTP

• In vivo and in vitro, transcription of the gal operon from the P2 promoter is very low when the quantity of UTP is high, and vice versa.
• Intriguingly, UTP modulates the phase of promoter clearing by RNA polymerase at the P2 promoter.
• In vitro, RNA polymerase clears poorly at P2 and generates a substantial number of abortive RNA oligomers, in contrast to P1.
• It additionally synthesises pseudo-templated RNA oligomers of the composition pppAUn (n=2to>20) at high doses of UTP because the enzyme “stutters” while adding uridine residues present at positions 3-5 of the P2 RNA and not the P1 RNA.
• At low UTP concentrations, RNA polymerase clears the promoter more efficiently and generates template-encoded regular gal RNA.
• The involvement of UTP in the synthesis of UDP sugars by the galactose pathway may explain why UTP controls the galactose pathway in a manner similar to how it controls its own synthesis in the pyrimidine operons.
• At high UTP concentrations, UDP galactose and UDP glucose levels are elevated and impede the synthesis of enzymes that produce them, notably galactose-1-phosphate uridyltransferase and uridine diphosphogalactose epimerase.

Repression by GalR and HU: DNA Looping

• GalR and HU inhibit transcription from the two gal promoters in a coordinated manner (negative control). The suppression requires the binding of GalR to two operators, Oe and O, which are dyad-symmetric 16-bp sequences.
• O e is positioned at position _60.5 upstream of the promoters, whereas O I is located at location +53.5 within the structural gene galE.
• In the presence of the cofactor HU (a heterodimer of two subunits, HUa and HUb), two operator-bound GalR molecules associate, resulting in the creation of a DNA loop enveloping the promoters.
• The DNA looping likely modifies the promoters’ structure, rendering them resistant to RNA polymerase caging.
• The nucleoprotein complex that represses gal promoters is referred to as the Gal repressosome because its creation involves the binding of a histone-like component of the bacterial nucleoid and a repressor.
• Despite the fact that GalR binds to Oe and Oi in the absence and presence of HU, GalR binding by itself does not result in DNA looping and the accompanying simultaneous suppression of the promoters.
• HU is essential for the effect; other histone-like proteins cannot be substituted. Despite the fact that HU is not a sequence-specific DNA-binding protein, a single molecule of HU heterodimer binds to and bends the gal DNA at a critical architectural point.
• Genetic study and modelling identified the interaction of GalR surfaces to produce a V-shaped tetrameric structure. Evaluation of DNA elastic energies favoured unambiguously a DNA loop in which OE and OI adopt an antiparallel orientation, resulting in DNA undertwisting.
• GalR and HU bind cooperatively because HU binding is dependent on GalR binding to both Oe and Oi, and GalR binding to the operators is increased by HU. Through a particular interaction between GalR and HU, HU is transported to the crucial DNA location by GalR.
• The entire procedure facilitates the contact between GalR and GalR, resulting in cooperation. The GalR-HU interaction may be transitory and was absent from the final configuration of the repressosome.
• The dependency of HU binding on GalR renders the HU-containing nucleoprotein complex that represses P1 and P2 transcriptionally responsive to the inducer D-galactose.
• In response to certain signals, this method makes DNA that has been “condensed” by binding proteins and rendered resistant to RNA polymerase action accessible for transcription.

Regulation in the Absence of DNA Looping: Interaction between GalR and RNA Polymerase

• Without DNA looping (i.e., in the absence of HU), occupancy of Oe by GalR alone represses P1 by a factor of four to five and activates P2 (twofold).
• The binding of O I has no impact on this dual control. P2 activation and P1 suppression are independent of one another.
• Even when both promoters are altered, GalR exerts its particular regulatory impact on one.
• The activation of P2 or suppression of P1 is not an inherent attribute of the promoter; the regulation can be reversed by reversing the angular orientation of the promoters relative to O e by inserting a 5-bp segment, or half a DNA helix, between OE and the promoters.
• Each promoter requires the development of a particular GalR-RNA polymerase-DNA complex for activation of P2 and repression of P1.
• GalR activates and represses by a direct contact with the promoter-bound RNA polymerases.

Activation of P2

• The activation of P2 by GalR coupled to DNA at position _60.5 (_55.5 relative to P2) is analogous to the stimulation of transcription of multiple promoters by activators that bind to DNA at position _60 and enhance open complex formation.
• The DNA-bound activator contacts an exposed segment (not necessarily the same segment) of aCTD to initiate transcription.
• A flexible hinge connects the aCTD to the remainder of the RNA polymerase molecule. Similar to these other systems, aCTD attaches to the area 40 bp upstream of P2 in P2.

Repression of P1

• GalR occupying DNA at position -60.5 represses P1 by contacting aCTD, which binds to P1 at position 45 bp upstream of P1.
• GalR suppresses RNA polymerase complex isomerization at P1.
• How interactions between the same two proteins result in opposing effects at the two promoters is still unknown. It is unknown why and under what conditions the dual behaviour of GalR toward P1 and P2 is initiated in cells in the absence of DNA looping.

Gal Isorepressor

• The gal operon is also controlled by an isorepressor (GalS).
• Although it does not appear that GalS represses the gal promoters via DNA looping, the isorepressor stimulates P2 and represses P1 in the same way that GalR does by binding to OE, albeit with lower effects.
• GalR and GalS regulate several additional operons, including those encoding galactose active transport systems with high and low affinity.
• The degree of control by GalR and GalS varies from operon to operon, possibly to coordinate galactose metabolism and facilitate effective transport across a broad range of galactose availability.

Properties of GalR and GalS

• The amino acid sequences of GalR and GalS are 85% similar. GalR and GalS are believed to feature two domains joined by a flexible hinge based on their similarity to proteins (GalR-LacI family) with known structures.
• In the amino domain of each subunit of GalR and GalS dimers, a helix-turn-helix motif recognises half of a dyad symmetry in OE and OI.
• The carboxy domains include the inducer binding sites, which have been identified through the identification and characterization of inducer-nonbinding (noninducible) repressor mutants, such as galRs, and through modelling.
• The process by which the inducer derepresses the P1 promoter under nonlooping conditions by binding to the -bound GalR has been extensively researched.
• Despite the fact that it has been demonstrated that inducer binding can detach GalR from the operator, P1 can be derepressed when O E is occupied by a GalR-inducer complex.
• These findings demonstrate that the separation of a repressor from an operator is not required for transcription. As the OE-bound GalR interacts with RNA polymerase post-binding to impede open complex formation at P1, the inducer functions by allosterically reducing the inhibitory contact between the proteins without dissociating the repressor from DNA.

Regulation by cAMP*CRP

• The global regulator cAMP*CRP complex has divergent effects on the gal promoters, like GalR and GalS; however, unlike GalR and GalS, the complex promotes transcription from P1 (three- to fourfold) and represses the same from P2 (10-fold).
• In contrast to a large group of promoters, including 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 in the gal operon achieves dual control by binding to DNA at position 41.5.
• It is suggested that cAMP*CRP represses P2 by preventing RNA polymerase from binding to the overlapping _35 region of the promoter, whereas the activation mechanism of P1 differs from that of the lac promoter.
• cAMP*CRP increases transcription initiation at P1 by enhancing RNA polymerase binding (closed-complex formation) and isomerization.
• A patch of amino acid residues (region 1) of the promoter distal subunit of the cAMP*CRP dimer interacts with an a-helix (helix 1) of the aCTD and aids in its separation from the N-terminal domain (aNTD) in order to bind to the area upstream of cAMP*CRP.
• The promoter-proximal subunit of cAMP*CRP interacts with a distinct portion of the aNTD via a separate patch of amino acid residues (termed region 2) The interaction with the aCTD increases RNA polymerase binding, whereas the connection with the aNTD drives isomerization.
• This demonstrates how the same regulatory protein can stimulate transcription initiation at two distinct biochemical phases by interacting with RNA polymerase in fundamentally different ways.
• However, the dual roles of cAMP*CRP allow the gal operon to be produced mostly from P1 in cAMP-sufficient cells and from P2 in cAMP-deficient (e.g., glucose-grown) cells.

References

• Lewis DE, Adhya S. Molecular Mechanisms of Transcription Initiation at gal Promoters and their Multi-Level Regulation by GalR, CRP and DNA Loop. Biomolecules. 2015 Oct 16;5(4):2782-807. doi: 10.3390/biom5042782. PMID: 26501343; PMCID: PMC4693257.
• Semsey, S., Virnik, K., & Adhya, S. (2006). Three-stage Regulation of the Amphibolic gal Operon: From Repressosome to GalR-free DNA. Journal of Molecular Biology, 358(2), 355–363. doi:10.1016/j.jmb.2006.02.022 
• Irani, M. H., Orosz, L., & Adhya, S. (1983). A control element within a structural gene: The gal operon of Escherichia coli. Cell, 32(3), 783–788. doi:10.1016/0092-8674(83)90064-8 
• https://en.wikipedia.org/wiki/Gal_operon
• https://www.slideshare.net/Divyapeddapalyam/galactose-operon-slide-share
• http://what-when-how.com/molecular-biology/gal-operon-molecular-biology/