Regulation
of Gene expression
Cells have mechanisms that allow them to
recognize specific environmental conditions under which they must either
activate or repress transcription of genes relevant under those conditions
allow them to turn the genes "on" or "off" as needed.
Control of gene expression can be exerted
at the level of
- Transcription (regulation
of RNA synthesis)
- RNA processing control
(regulation of intron/exon splicing)
- Translation (regulation
protein synthesis)
- Protein/enzyme function
(modulation of protein function)
Gene
regulation in prokaryotes
Gene expression is the most fundamental
level at which the genotype gives rise to the phenotype. Regulation of gene expression includes a wide
range of mechanisms that are used by cells to increase or decrease the
production of specific gene products (protein or RNA), and is informally termed
gene regulation. Bacteria are exposed to
an ever-changing environment in which nutrient availability may increase or
decrease radically. Bacteria respond to such variations in their environment by
altering their gene expression pattern; thus, they express different enzymes
depending on the carbon sources and other nutrients available to them. The process of transcription, which is the
synthesis of RNA from a DNA template, is where the regulation of the gene
expression is most likely to occur.
A
constitutive gene or a housekeeping
gene is a gene that is transcribed continually at a relatively constant
level. The housekeeping gene's products are typically needed for maintenance of
the cell. Examples are genes that code
for components of ribosome, enzymes needed for glucose metabolism, protein
synthesis, etc. An
inducible gene or regulated gene is a gene whose expression is
responsive to environmental change and is transcribed only when needed. Example is the enzymes needed for utilisation
of lactose in bacteria.
Bacterial genes are organized into operons, or clusters of coregulated
genes. In addition to being physically close in the genome, these genes are
regulated such that they are all turned on or off together. operon is a
functioning unit of genomic DNA containing a cluster of genes under the control
of a single promoter. The genes
contained in the operon are either expressed together or not at all. Grouping related genes under a common control
mechanism allows bacteria to rapidly adapt to changes in the environment.
An operon is made up of 3 basic DNA
components:
- Promoter – a nucleotide sequence that is recognized by RNA polymerase, which then initiates transcription.
- Operator – a segment of DNA that a repressor binds to. In the lac operon it is a segment between the promoter and the genes of the operon.
- Structural
genes – the genes that are
co-regulated by the operon. An operon contains one or more structural genes
which are transcribed into one polycistronic mRNA (a single mRNA molecule that
codes for more than one protein).
Operon
regulation can be either negative or positive by
induction or repression
Negative
control involves the binding of a repressor to
the operator to prevent transcription.
In negative inducible operons, transcription is blocked by a
regulatory repressor protein bound to the operator. If an inducer molecule is
present, it binds to the repressor and changes its conformation so that it is
unable to bind to the operator. This allows expression of the operon. The lac
operon is a negatively controlled inducible operon, where the inducer molecule
is lactose.
In negative repressible operons, transcription of the operon normally
takes place. Repressor proteins are produced by a regulator gene, but they are
unable to bind to the operator in their normal conformation. When certain
molecules called corepressors are bound by the repressor protein, repressor
becomes active. The activated repressor protein binds to the operator and
prevents transcription. The trp operon, involved in the synthesis of
tryptophan, is a negatively controlled repressible operon.
With positive
control, an activator protein stimulates transcription by binding to DNA.
In
positive inducible operons,
activator proteins are normally unable to bind to the DNA. When an inducer is
bound by the activator protein, it undergoes a change in conformation so that
it can bind to the DNA and activate transcription.
In positive repressible operons, the activator proteins are normally
bound to the DNA segment. However, when an inhibitor is bound by the activator,
it is prevented from binding the DNA. This stops activation and transcription
of the system.
Lac
operon (lactose operon)
This operon is required for the transport
and metabolism of lactose in E coli
and many other enteric bacteria. Although glucose is the preferred carbon
source, the lac operon allows for the utilisation of lactose when glucose is
not available.
The lac operon consists of three
structural genes, promoter, terminator, regulator, and an operator.
o
P strands for promoter;
it is the site where RNA polymerase attaches in order to transcribe mRNA.
o
The
I gene is called a regulator
gene; it is transcribed to make mRNA that translat to a repressor protein.
o
O
stands for Operator; short sequence of bases that can be recognized by
repressor protein (acts like a switch).
o
Z, Y and A
are structural genes.
- lacZ encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose.
- lacY encodes lactose permease (LacY), a transmembrane symporter that pumps β-galactosides into the cell using a proton gradient in the same direction.
- lacA encodes galactoside O-acetyltransferase (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.
The lac operon is under both negative and positive
control.
Negative Control of Transcription:
When lactose is absent
When lactose is present
The I mRNA is translated into repressor protein. Lactose binds to the repressor and converts
the repressor into an inactive state, where it cannot bind the Operator. RNA polymerase binds to the promoter and
several copies of ZYA mRNA is made. Expression
of permease causes more lactose to enter the cell and the
operon will remain fully active until the lactose is no longer present
Positive Control of Transcription:
Cells
use glucose as carbon source, even if alternative sources like lactose are
present. Therefore the lac operon is expressed at a high level only when
glucose absent. Glucose is always
metabolized first in preference to other sugars. Only after glucose is
completely utilized is lactose degraded. The lac operon is repressed even
though lactose (the inducer) is present. The ecological rationale is that
glucose is a better source of energy than lactose since its utilization
requires two less enzymes. Since the availability of glucose represses the
enzymes for lactose utilization, this type of repression became known as
catabolite repression or the glucose effect.
RNA polymerase cannot bind to promoter without the aid of CAP-cAMP
complex.
When glucose is present and lactose is present
In the presence of glucose, adenylate cyclase (AC)
activity is blocked. AC is required to synthesize cAMP from ATP. Therefore,
cAMP levels are low, CAP is inactive and transcription does not occur.
When glucose is absent and lactose is present
In
the absence of glucose, adenylate cyclase (AC) activity is not blocked, cAMP
levels are high, CAP is activated by cAMP, and transcription occurs (in the
presence of lactose since it is the inducer).
_______________________________________________________
The trp operon
This
operon involves a group of genes that are transcribed together and codes for
the components for production of tryptophan. The trp operon is present in many
bacteria, and was first characterized in Escherichia
coli.
The
trp operon of E. coli controls the biosynthesis of tryptophan in the cell from
the initial precursor chorismic acid. This operon contains genes for the
production of five proteins (A, B, C, D and E) which are used to produce three
enzymes. The products of the E and D genes form a multimeric protein to
produce the enzyme anthranilate synthetase. This enzyme catalyzes the
first two reactions in the tryptophan pathway. The next enzyme is indole
glycerol phosphate synthetase, the product of the C locus. The final
step in the reaction produces tryptophan catalyzed by tryptophan synthetase,
an enzyme that is a multimer of two proteins (product of the B and A
genes).
The
trp operon consists of repressor, promoter, operator and the structural genes. Unlike
the lac operon, the gene for the repressor is located in another part of the E.
coli genome. Another difference is that the operator resides entirely within
the promoter.
- When there's an excess of trp in the cell, some of it will bind to and activate a free-floating regulator protein (R).
- The R-trp complex has a high affinity for the trp operator. When it is attached, it blocks the attachment of RNA polymerase, stopping transcription. The operon is thus turned "off". The R protein is a repressor.
- Because the product of the operon (the amino acid tryptophan) is involved in repressing the very gene responsible for its manufacture, tryptophan is said to be a corepressor
- When the cell's trp concentration is very low, trp will diffuse off the regulator protein. The naked regulator loses its affinity for the operator, and detaches. RNA polymerase can attach to the operon, transcribing the five genes coding for the tryptophan-manufacturing enzymes
- In this condition, the operon is said to be derepressed until trp concentration raises enough to form active R-TRP, which will again repress the operon.
In prokaryotes, no nuclear membrane
separates transcription and translation and the ribosomes will bind the RNA
soon after it emerges from the RNA polymerase. The close linkage of the
processes can lead to interdependent control mechanisms such as the attenuation. Attenuation is a regulatory feature found throughout Archaea and
Bacteria causing premature termination of transcription. Since in
bacteria, transcription and translation are coupled, attenuation effectively
blocks gene expression. Attenuation involves a stop signal (attenuator) located
in the DNA segment that corresponds to the leader sequence of mRNA. During
attenuation, the ribosome becomes stalled (delayed) in the attenuator region in
the mRNA leader.
When
gene transcription is moderated by the attachment of a protein to the operator,
the gene/operon system is said to be operating under operator control. The trp operon can function also under
attenuator control. Unlike repression,
attenuation is effected at the level of translation.
In trp operon attenuation is controlled by the trp leader sequence.
The
transcript of the trp operon includes 162 nucleotides upstream of the initiation
codon for trpE (the first structural gene). Leader sequence contains four complementary
regions that can bind together in various ways to form stem loops. This leader
mRNA also includes a section encoding a leader peptide of 14 amino acids. Leader peptide contains two adjacent
tryptophan (trp) molecules.
If
tryptophan is present, the sensor peptide is easily made and the trp mRNA is
NOT made.
If
tryptophan is scarce, the leader peptide is not made and the full operon is
transcribed then translated into tryptophan synthetic enzymes.
- The leader sequence immediately 5' of the trpE gene controls the expression of the operon through a process called attenuation.
- This sequence has four domains (1-4) capable of base pairing in various combinations to form hairpin structures.
- Attenuation depends upon the ability of regions 1 and 2 and regions 3 and 4 of the trp leader sequence to base pair and form hairpin secondary structures
- Domain 3 of the mRNA can base pair with either domain 2 or domain 4.
- A part of the leader mRNA containing regions 3 and 4 and a string of eight U's is called the attenuator. The region 3+4 hairpin structure acts as a transcription termination signal; as soon as it forms, the RNA and the RNA polymerase are released from the DNA.
- Two adjacent tryptophan (trp) codons within the leader mRNA sequence are essential in the operon's regulation. The speed with which the ribosome travels along the trp transcript depends on the availability of trp.
During periods of tryptophan scarcity,
- A ribosome translating the coding sequence for the leader peptide may stall when it encounters the two tryptophan (trp) codons because of the shortage of tryptophan-carrying tRNA molecules.
- Because a stalled ribosome at this site blocks region 1, a region 1+2 hairpin cannot form and region 2+3 hairpin is formed.
-
When tryptophan is readily available,
- A
ribosome can complete translation of the leader peptide without stalling.
- As
it pauses at the stop codon, it blocks region 2, preventing it from base
pairing.
- As a result, the region 3+4 structure forms and terminates transcription near the end of the leader sequence and the structural genes of the operon are not transcribed (nor translated).
This
is example of a "riboswitch",
a mechanism which can control transcription and translation through
interactions of molecules with in an mRNA
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