The gal operon in E. coli encodes enzymes essential for galactose metabolism and is regulated by various mechanisms, including the binding of repressor molecules to specific operators, which inhibits transcription. Key enzymes are involved in both the catabolism of D-galactose and the synthesis of precursors for complex carbohydrates. The operon's structure and regulation allow for coordinated gene expression, although mutations in the promoter can disrupt the functionality of all associated genes.

1. Galactose operon
Divya peddapalyam(Msc.Biotechnology)
Garden city University
Bengaluru;Karnataka;India

2. Introduction:
The gal operon is a prokaryotic operon, which encodes enzymes necessary for galactose metabolism. The operon contains
two operators, OE (for external) and OI.
Repression of gene expression works via binding of repressor molecules to the two operators. These repressors dimerize, creating a loop in
the DNA. The loop as well as hindrance from the external operator prevent RNA polymerase from binding to the promoter, and thus
prevent transcription.
The gal operon of E. coli consists of 3 structural genes: galE (epimerase), galT (galactose transferase), and galK (galactokinase), which are
transcribed from two overlapping promoters PG1 and PG2 upstream from galE.
Regulation of the operon is complex since the GalE product, an epimerase that converts UDP-glucose into UDP-galactose, is required for the
formation of UDP-galactose for cell wall biosynthesis, in particular the cell wall component lipopolysaccharide, even when cells are not
using galactose as a carbon/energy source.
The gal operon is controlled by CRP-cAMP as for the lac operon .CRP-cAMP binds to -35 region promoting transcription from PG1 but
inhibiting transcription from PG2. When cells are grown in glucose, basal level transcription occurs from PG2. The unlinked galRgene encodes
the repressor for this system. A tetrameric GalR repressor binds to 2 operators, one located at +55 and one located at -60 relative to the PG1
start site. Looping of the DNA blocks the access of RNA polymerase to promoters and/or inhibits formation of the open complex. When GalR
binds as a dimer to the -60 site only, promoter PG2 is activated, not repressed, allowing basal levels of GalE to be produced. In this state
promoter PG1 is inactivated through interactions with the alpha subunit of RNA polymerase

3. Leloir pathway of D-Galactose metabolism
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. The
galactose metabolizing enzymes 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 . Thus, aldose-1-epimerase links the enzymes of
lactose and galactose metabolism into a common pathway

4. Schematic representation of galactose operon
A schematic presentation of the galactose operon. The -10 regions of the P1 and P2 promoters are shown in
boxes. The trans- cription initiation sites are indicated by arrows and underlined. CRP points to its DNA binding
site. O E and O I designate the op- erators where GalR binds. HU protein binds to hbs . The transcrip- tion
initiation site of the P1 promoter is assigned as +1, Nucleo- tide positions of the operon, thus, have been
assigned relative to the P1 initiation site.

5. Advantages and disadvantages
• Operon, genetic regulatory system found in bacteria and their viruses in which genes coding for
functionally related proteins are clustered along the DNA. This feature allows protein synthesis to be
controlled coordinately in response to the needs of the cell. By providing the means to produce proteins
only when and where they are required, the operon allows the cell to conserve energy (which is an
important part of an organism’s life strategy).
• A typical operon consists of a group of structural genes that code for enzymes involved in a metabolic
pathway, such as the biosynthesis of an amino acid. These genes are located contiguously on a stretch of
DNA and are under the control of one promoter (a short segment of DNA to which the RNA polymerase
binds to initiate transcription). A single unit of messenger RNA (mRNA) is transcribed from the operon and
is subsequently translated into separate proteins.
• The promoter is controlled by various regulatory elements that respond to environmental cues. One
common method of regulation is carried out by a regulator protein that binds to the operator region, which
is another short segment of DNA found between the promoter and the structural genes. The regulator
protein can either block transcription, in which case it is referred to as a repressor protein; or as an activator
protein it can stimulate transcription. Further regulation occurs in some operons: a molecule called
an inducer can bind to the repressor, inactivating it; or a repressor may not be able to bind to the operator
unless it is bound to another molecule, the corepressor. Some operons are under attenuator control, in which
transcription is initiated but is halted before the mRNA is transcribed. This introductory region of the
mRNA is called the leader sequence; it includes the attenuator region, which can fold back on itself,
forming a stem-and-loop structure that blocks the RNA polymerase from advancing along the DNA.
• While an operon provides the advantage of being able to initiate transcription at one point and transcribe
many genes, it has its disadvantages as well. One disadvantage is that if the promoter for the operon
sequence is mutated, all the genes in the operon cannot be transcribed.

6. Review article:
Results:
Effect of carbon source on the rates of UDP-Gal epimerase, galactokinase, and /8-galactosidase syntheses. Effects
of carbon source and cAMP on the constitutive rates of UDP-Gal epimerase and galactokinase syntheses.Along
with The galactose operon of E. coli consists of three structural genes, galE, galT, and galK, which code for a
UDP-Gal epimerase, a uridyl transferase, and a galactokinase, respectively. The operon is negatively controlled by
a repressor protein, the product of the galR gene, which is not linked to the gal operon (4). As we wanted to
distinguish effects exerted at the level of transcription initiation from effects on premature transcription
termination (polar effects), in all our experiments we measured the rates of synthesis of UDP-Gal epimerase (the
promoterproximal enzyme) and galactokinase (the promoter-distal enzyme).

9. Results:
Specific enzymatic activity of galactose enzymes in galO
constitutive mutants grown in minimal glucose medium.
Specific enzymatic activity of galactokinase in galO
constitutive mutants grown in minimal glycerol medium

10. Results:

11. Schematic of the strategy used
to clone the S. lividans galK
gene.

12. Identification of galactokinase produced by S. lividans. Cells
were grown in SLAB. Hybridization analysis of S. lividans
chromosomal DNA
Restriction map of the chromosomal insert containing the
S. lividans gal operon

13. Comparison of the E. coli, S. lividans, and Saccharomyces carlbergensis gal gene products.

14. Bioinformatic studies:
• single point mutation at -10 box in the galK promoter can significantly affect the expression of gal operon
and is largely responsible for the Gal+ phenotype of S. thermophilus.