lac operon

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The lac operon is an operon required for the transport and metabolism of lactose in
Escherichia coli and some other enteric bacteria. It consists of three adjacent
structural genes, a promoter, a terminator, and an operator. The lac operon is
regulated by several factors including the availability of glucose and of lactose.
Gene regulation of the lac operon was the first complex genetic regulatory
mechanism to be elucidated and is one of the foremost examples of prokaryotic
gene regulation.

In its natural environment, the lac operon is a complex mechanism to digest lactose
efficiently. The cell can use lactose as an energy source by producing the enzyme β-
galactosidase to digest that lactose into glucose. However, it would be inefficient to
produce enzymes when there is no lactose available, or if there is a more readily-
available energy source available such as glucose. The lac operon uses a two-part
control mechanism to ensure that the cell expends energy producing β-
galactosidase, β-galactoside permease and thiogalactoside transacetylase (also
known as galactoside O-acetyltransferase) only when necessary. It achieves this
with the lac repressor, which halts production in the absence of lactose, and the
Catabolite activator protein (CAP), which assists in production in the absence of
glucose. This dual control mechanism causes the sequential utilization of glucose
and lactose in two distinct growth phases, known as diauxie. Similar diauxic growth
patterns have been observed in bacterial growth on mixtures of other sugars as
well, such as mixtures of glucose and xylose, or of glucose and arabinose, etc. The
genetic control mechanisms underlying such diauxic growth patterns are known as
xyl operon and ara operon, etc.Contents [hide]

1 Structure of the lac operon

1.1 Genetic nomenclature

2 Lactose analogues

3 Classification of regulatory mutants

4 Regulation by cyclic AMP

5 Multimeric nature of repressor and the complex operator

5.1 Mechanism of induction

6 Use in molecular biology

7 References

8 External links

[edit]

Structure of the lac operon

Structure of lactose and the products of its cleavage.

The lac operon consists of three structural genes, and a promoter, a terminator,
regulator, and an operator. The three structural genes are: lacZ, lacY, and lacA.

lacZ encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the

disaccharide lactose into glucose and galactose.

lacY encodes β-galactoside permease (LacY), a membrane-bound transport protein

that pumps lactose into the cell.

lacA encodes β-galactoside transacetylase (LacA), an enzyme that transfers an

acetyl group from acetyl-CoA to β-galactosides.

Only lacZ and lacY appear to be necessary for lactose catabolism.

Specific control of the lac genes depends on the availability of the substrate lactose
to the bacterium. The proteins are not produced by the bacterium when lactose is
unavailable as a carbon source. The lac genes are organized into an operon; that is,
they are oriented in the same direction immediately adjacent on the chromosome
and are co-transcribed into a single polycistronic mRNA molecule. Transcription of
all genes starts with the binding of the enzyme RNA polymerase (RNAP), a DNA-
binding protein, which binds to a specific DNA binding site, the promoter,
immediately upstream of the genes. From this position RNAP proceeds to transcribe
all three genes (lacZYA) into mRNA. The DNA sequence of the E. coli lac operon, the
lacZYA mRNA, and the lacI genes are available from GenBank (view).
The first control mechanism is the regulatory response to lactose, which uses an
intracellular regulatory protein called the lactose repressor to hinder production of
β-galactosidase in the absence of lactose. The lacI gene coding for the repressor lies
nearby the lac operon and is always expressed (constitutive). If lactose is missing
from the growth medium, the repressor binds very tightly to a short DNA sequence
just downstream of the promoter near the beginning of lacZ called the lac operator.
The repressor binding to the operator interferes with binding of RNAP to the
promoter, and therefore mRNA encoding LacZ and LacY is only made at very low
levels. When cells are grown in the presence of lactose, however, a lactose
metabolite called allolactose, which is a recombination of glucose and galactose,
binds to the repressor, causing a change in its shape. Thus altered, the repressor is
unable to bind to the operator, allowing RNAP to transcribe the lac genes and
thereby leading to high levels of the encoded proteins.

The second control mechanism is a response to glucose, which uses the Catabolite
activator protein (CAP) to greatly increase production of β-galactosidase in the
absence of glucose. Cyclic adenosine monophosphate (cAMP) is a signal molecule
whose prevalence is inversely proportional to that of glucose. It binds to the CAP,
which in turn allows the CAP to bind to the CAP promoter (on the left in the diagram
below), which assists the RNAP in binding to the DNA. In the absence of glucose, the
prevalence of cAMP and binding of the CAP to the DNA significantly increases the
production of β-galactosidase, enabling the cell to digest the lactose needed to
produce glucose.

The diagram below summarizes these statements.

lac operon in detail

[edit]

Genetic nomenclature

Three-letter abbreviations are used to describe phenotypes in bacteria including E.

coli.

Examples include:

Lac (the ability to use lactose),

His (the ability to synthesize the amino acid histidine)

Mot (swimming motility)

SmR (response to the antibiotic streptomycin)

In the case of Lac, wild type cells are Lac+ and are able to use lactose as a carbon
and energy source, while Lac- mutant derivatives cannot use lactose. The same
three letters are typically used (lower-case, italicized) to label the genes involved in
a particular phenotype, where each different gene is additionally distinguished by
an extra letter. The lac genes encoding enzymes are lacZ, lacY, and lacA. The fourth
lac gene is lacI, encoding the lactose repressor—"I" stands for inducibility.

One may distinguish between structural genes encoding enzymes, and regulatory
genes encoding proteins that affect gene expression. Current usage expands the
phenotypic nomenclature to apply to proteins: thus, LacZ is the protein product of
the lacZ gene, β-galactosidase. Various short sequences that are not genes also
affect gene expression, including the lac promoter, lac p, and the lac operator, lac
o. Although it is not strictly standard usage, mutations affecting lac o are referred to
as lac oc, for historical reasons.

[edit]

Lactose analogues

IPTG

ONPG

X-gal

allolactose
A number of lactose derivatives or analogs have been described that are useful for
work with the lac operon. These compounds are mainly substituted galactosides,
where the glucose moiety of lactose is replaced by another chemical group.

Isopropyl-β-D-thio-galactoside (IPTG) is frequently used as an inducer of the lac

operon for physiological work.[1] IPTG binds to repressor and inactivates it, but is
not a substrate for β-galactosidase. One advantage of IPTG for in vivo studies is that
since it cannot be metabolized by E. coli its concentration remains constant and the
rate of expression of lac p/o-controlled genes, is not a variable in the experiment.
IPTG intake is dependent on the action of lactose permease.[1]

Phenyl-β-D-galactose (phenyl-Gal) is a substrate for β-galactosidase, but does not

inactivate repressor and so is not an inducer. Since wild type cells produce very
little β-galactosidase, they cannot grow on phenyl-Gal as a carbon and energy
source. Mutants lacking repressor are able to grow on phenyl-Gal. Thus, minimal
medium containing only phenyl-Gal as a source of carbon and energy is selective
for repressor mutants and operator mutants. If 108 cells of a wild type strain are
plated on agar plates containing phenyl-Gal, the rare colonies which grow are
mainly spontaneous mutants affecting the repressor. The relative distribution of
repressor and operator mutants is affected by the target size. Since the lacI gene
encoding repressor is about 50 times larger than the operator, repressor mutants
predominate in the selection.

Other compounds serve as colorful indicators of β-galactosidase activity.

ONPG is cleaved to produce the intensely yellow compound, orthonitrophenol, and

is commonly used as a substrate for assay of β-galactosidase in vitro. [2]

Colonies that produce β-galactosidase are turned blue by X-gal (5-bromo-4-chloro-3-

indolyl-β-D-galactoside). [3]

Allolactose is an isomer of lactose and is the inducer of the lac operon. Lactose is
galactose-(β1->4)-glucose, whereas allolactose is galactose-(β1->6)-glucose.
Lactose is converted to allolactose by β-galactosidase in an alternative reaction to
the hydrolytic one. A physiological experiment which demonstrates the role of LacZ
in production of the "true" inducer in E. coli cells is the observation that a null
mutant of lacZ can still produce LacY permease when grown with IPTG but not when
grown with lactose. The explanation is that processing of lactose to allolactose
(catalyzed by β-galactosidase) is needed to produce the inducer inside the cell.

[edit]

Classification of regulatory mutants

A conceptual breakthrough of Jacob and Monod[4] was to recognize the distinction
between regulatory substances and sites where they act to change gene
expression. A former soldier, Jacob used the analogy of a bomber that would release
its lethal cargo upon receipt of a special radio transmission or signal. A working
system requires both a ground transmitter and a receiver in the airplane. Now,
suppose that the usual transmitter is broken. This system can be made to work by
introduction of a second, functional transmitter. In contrast, he said, consider a
bomber with a defective receiver. The behavior of this bomber cannot be changed
by introduction of a second, functional aeroplane.

To analyze regulatory mutants of the lac operon, Jacob developed a system by

which a second copy of the lac genes (lacI with its promoter, and lacZYA with
promoter and operator) could be introduced into a single cell. A culture of such
bacteria, which are diploid for the lac genes but otherwise normal, is then tested for
the regulatory phenotype. In particular, it is determined whether LacZ and LacY are
made even in the absence of IPTG. This experiment, in which genes or gene clusters
are tested pairwise, is called a complementation test.

This test is illustrated in the figure (lacA is omitted for simplicity). First, certain
haploid states are shown (i.e. the cell carries only a single copy of the lac genes).
Panel (a) shows repression, (b) shows induction by IPTG, and (c) and (d) show the
effect of a mutation to the lacI gene or to the operator, respectively. In panel (e) the
complementation test for repressor is shown. If one copy of the lac genes carries a
mutation in lacI, but the second copy is wild type for lacI, the resulting phenotype is
normal---but lacZ is expressed when exposed to inducer IPTG. Mutations affecting
repressor are said to be recessive to wild type (and that wild type is dominant), and
this is explained by the fact that repressor is a small protein which can diffuse in the
cell. The copy of the lac operon adjacent to the defective lacI gene is effectively
shut off by protein produced from the second copy of lacI.

If the same experiment is carried out using an operator mutation, a different result
is obtained (panel (f)). The phenotype of a cell carrying one mutant and one wild
type operator site is that LacZ and LacY are produced even in the absence of the
inducer IPTG; because the damaged operator site, does not permit binding of the
repressor to inhibit transcription of the structural genes. The operator mutation is
dominant. When the operator site where repressor must bind is damaged by
mutation, the presence of a second functional site in the same cell makes no
difference to expression of genes controlled by the mutant site.
A more sophisticated version of this experiment uses marked operons to distinguish
between the two copies of the lac genes and show that the unregulated gene(s) are
the ones next to the mutant operator (panel (g). For example, suppose that one
copy is marked by a mutation inactivating lacZ so that it can only produce the LacY
protein, while the second copy carries a mutation affecting lacY and can only
produce LacZ. In this version, only the copy of the lac operon that is adjacent to the
mutant operator is expressed without IPTG. We say that the operator mutation is
cis-dominant, it is dominant to wild type but affects only the copy of the operon
which is immediately adjacent to it.

This explanation is misleading in an important sense, because it proceeds from a

description of the experiment and then explains the results in terms of a model. But
in fact, it is often true that the model comes first, and an experiment is fashioned
specifically to test the model. Jacob and Monod first imagined that there must be a
site in DNA with the properties of the operator, and then designed their
complementation tests to show this.

The dominance of operator mutants also suggests a procedure to select them

specifically. If regulatory mutants are selected from a culture of wild type using
phenyl-Gal, as described above, operator mutations are rare compared to repressor
mutants because the target-size is so small. But if instead we start with a strain
which carries two copies of the whole lac region (that is diploid for lac), the
repressor mutations (which still occur) are not recovered because complementation
by the second, wild type lacI gene confers a wild type phenotype. In contrast,
mutation of one copy of the operator confers a mutant phenotype because it is
dominant to the second, wild type copy.

[edit]

Regulation by cyclic AMP This section does not cite any references or sources.

Please help improve this article by adding citations to reliable sources. Unsourced
material may be challenged and removed. (May 2009)

The experimental microorganism used by François Jacob and Jacques Monod was
the common laboratory bacterium, E. coli, but many of the basic regulatory
concepts that were discovered by Jacob and Monod are fundamental to cellular
regulation in organisms. The key idea is that proteins are not synthesized when
they are not needed--- E. coli conserves cellular resources and energy by not
making the three Lac proteins when there is no need to metabolize lactose, such as
when other sugars like glucose are available. The following section discusses how E.
coli controls certain genes in response to metabolic needs.

During World War II, Monod was testing the effects of combinations of sugars as
nutrient sources for E. coli. He found that bacteria grown with two different sugars
often displayed two phases of growth. For example, if glucose and lactose were
both provided, glucose would be metabolized first (growth phase I, see Figure 2)
and then lactose (growth phase II). This phenomenon is called diauxie.

Figure 2: Monod's "bi-phasic" growth curve

Metabolism of lactose does not occur during the first part of the diauxic growth
curve because β-galactosidase is not made when both glucose and lactose are
present in the medium.

Explanation of this depended on the characterization of additional mutations

affecting the lac genes other than those explained by the classical model. Two other
genes, cya and crp, were known that map far away from lac, and when mutant
result in a decreased level of expression in the presence of IPTG and even in a
strain which is mutant for repressor or operator. The discovery of cyclic AMP in 1957
(in eukaryotic cells) and a decade later in E. coli led to the demonstration that
mutants defective in one of these genes could be restored to full activity by the
addition of cyclic AMP to the medium.

The cya gene encodes adenylate cyclase, which produces cyclic AMP. In a cya
mutant, the absence of cyclic AMP makes the expression of the lacZYA genes more
than ten times lower than normal. Addition of cyclic AMP corrects the low Lac
expression characteristic of cya mutants. The second gene, crp, encodes a protein
called catabolite activator protein (CAP) or cAMP receptor protein (CRP).

This dual regulation causes the lactose metabolism enzymes to be made in small
quantities in the presence of both glucose and lactose (sometimes called leaky
expression) due to lactose inhibiting LacI from binding to the operator, but at high
cAMP concentrations and in the presence of lactose there are high levels of
expression (Phase II in Figure 2). Leaky expression is necessary in order to allow for
metabolism of some lactose after the glucose source is expended, but before lac
expression is fully activated.

In summary:

When lactose is absent then there is very little Lac enzyme production (the operator
has LacI bound to it).

When lactose is present but a preferred carbon source (like glucose) is also present
then a small amount of enzyme is produced (LacI is not bound to the operator).

When lactose is the favoured carbon source (for example in the absence of glucose)
cAMP-CAP binds upstream of the promoter at a specific site. This bends the DNA
around the protein which creates tension, and allows the RNA polymerase to bind to
the promoter and Lac enzyme production is maximised. The DNA is not easily
unwound under normal conditions, without the bound CAP, as the DNA contains a
large number of the nucleotides which have 3 hydrogen bonds between them,
needing more energy to part them[citation needed]

{{ The delay between growth phases reflects the time needed to produce sufficient
quantities of lactose-metabolizing enzymes. First, the CAP regulatory protein has to
assemble on the lac promotor, resulting in an increase in the production of lac
mRNA. More available copies of the lac mRNA results in the production (see
translation) of significantly more copies of LacZ (β-galactosidase, for lactose
metabolism) and LacY (lactose permease to transport lactose into the cell). After a
delay needed to increase the level of the lactose metabolizing enzymes, the
bacteria enter into a new rapid phase of cell growth.

Two puzzles of catabolite repression relate to how cAMP level is actually coupled to
the presence of glucose, and secondly, why the cells should even bother. After
lactose is cleaved it actually forms glucose and galactose (easily converted to
glucose). In metabolic terms, lactose is just as good a carbon and energy source as
glucose. The cAMP level is related not to intracellular glucose concentration but to
the rate of glucose transport, which influences the activity of adenylate cyclase. (In
addition, glucose transport also leads to direct inhibition of the lactose permease.)
As to why E. coli works this way, one can only speculate. All enteric bacteria
ferment glucose, which suggests they encounter it frequently. It is possible that a
small difference in efficiency of transport or metabolism of glucose v. lactose makes
it advantageous for cells to regulate the lac operon in this way [5].
[edit]

Multimeric nature of repressor and the complex operator

The lac repressor is a tetramer of identical subunits. Each subunit contains a helix-
turn-helix (HTH) motif capable of binding to DNA. The operator site where repressor
binds is a DNA sequence with inverted repeat symmetry. The two DNA half-sites of
the operator together bind to two of the subunits of the tetrameric repressor.
Although the other two subunits of repressor are not doing anything in this model,
this property was not understood for many years.

Eventually it was discovered that two additional (minor) operators are involved in
lac regulation. One (O3) lies in the end of the lacI gene and the other (O2) is about
400 bp downstream in the early part of lacZ. These two sites were not found in the
early work because they have redundant functions and individual mutations do not
affect repression very much. Single mutations to either O2 or O3 have only 2 to 3-
fold effects. However, their importance is demonstrated by the fact that a double
mutant defective in both O2 and O3 is dramatically de-repressed (by about 70-fold).

In the current model, repressor is bound simultaneously to both the main operator
O1 and to either O2 or O3. The intervening DNA loops out from the complex. The
redundant nature of the two minor operators suggests that it is not a specific looped
complex that is important. One idea is that the system works through tethering. If
bound repressor releases from O1 momentarily, binding to a minor operator keeps
it in the vicinity, so that it may rebind quickly. This would increase the affinity of
repressor for O1.

[edit]

Mechanism of induction

The repressor is an allosteric protein, i.e. it can assume either one of two slightly
different shapes, which are in equilibrium with each other. In one form the repressor
is capable of binding to the operator DNA, and in the other form it cannot bind to
the operator. According to the classical model of induction, binding of the inducer,
either allolactose or IPTG, to the repressor affects the distribution of repressor
between the two shapes. Thus, repressor with inducer bound is stabilized in the
non-DNA-binding conformation. However, this simple model cannot be the whole
story, because repressor is bound quite stably to DNA, yet it is released rapidly by
addition of inducer. Therefore it seems clear that repressor can also bind inducer
while still bound to DNA. It is still not entirely known what the exact mechanism of
binding is.

[edit]

Use in molecular biology

The lac gene and its derivatives are amenable to use as a reporter gene in a
number of bacterial-based selection techniques such as two hybrid analysis, in
which the successful binding of a transcriptional activator to a specific promoter
sequence must be determined.[3] In LB plates containing X-gal, the colour change
from white colonies to a shade of blue, corresponds to about 20-100 β-
galactosidase units, while tetrazolium lactose and MacConkey lactose media have a
range of 100-1000 units, being most sensitive in the high and low parts of this
range respectively. [3] Since MacConkey lactose and tetrazolium lactose media
both rely on the products of lactose breakdown, they require the presence of both
lacZ and lacY genes. The many lac fusion techniques which include only the lacZ
gene are thus suited to the X-gal plates [3] or ONPG liquid broths [6]

[edit]

References This article needs additional citations for verification.

Please help improve this article by adding reliable references. Unsourced material
may be challenged and removed. (December 2007)

^ Hansen LH, Knudsen S, Sørensen SJ (June 1998). "The effect of the lacY gene on
the induction of IPTG inducible promoters, studied in Escherichia coli and
Pseudomonas fluorescens". Curr. Microbiol. 36 (6): 341–7.
doi:10.1007/s002849900320. PMID 9608745.

^ "ONPG (β-Galactosidase) test". September 2000.

^ a b c d Joung J, Ramm E, Pabo C (2000). "A bacterial two-hybrid selection system

for studying protein-DNA and protein-protein interactions". Proc Natl Acad Sci USA
97 (13): 7382–7. doi:10.1073/pnas.110149297. PMID 10852947.

^ Jacob F; Monod J (June 1961). "Genetic regulatory mechanisms in the synthesis of

proteins". J Mol Biol. 3: 318–56. PMID 13718526.

^ Vazquez A, Beg QK, Demenezes MA, et al (2008). "Impact of the solvent capacity
constraint on E. coli metabolism". BMC Syst Biol 2: 7. doi:10.1186/1752-0509-2-7.
PMID 18215292.
^ "OPNG (β-galactosidase) test". Standards Unit, Evaluations and Standards
Laboratory Centre for Infections (UK).

[edit]

External links

MeSH Lac+Operon

Virtual Cell Animation Collection Introducing: The Lac Operon

lac operon on www.ecolicommunity.org/EcoliWiki, the community annotation

system of EcoliHub.

Staining Whole Mouse Embryos for β-Galactosidase (lacZ) Activity

EcoliWiki Page: LacZ

Mcgraw hill animation for Lac Operon[hide]

v•d•e

Transcription (Prokaryotic, Eukaryotic)

Promoter (Pribnow box, TATA box, BRE, CAAT box) - Operon (gal operon, lac operon,
trp operon) - Terminator - Enhancer - Insulator - Repressor (lac repressor, trp
repressor) - Silencer - Histone methylation

Categories: Gene expression

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