GENE EXPRESSION

PROKARYOTES

GENE: Gene Expression in Prokaryotes:

A gene is the basic physical and Prokaryotes only transcribe genes that their end-
functional unit of heredity. proteins are needed at the time. They do this in order
Genes, which are made up of to save up energy and increase efficiency. The regula-
DNA, act as instructions to tion of gene expression is depended mainly on their
make molecules called proteins. immediate environment, for example on the presence
In humans, genes vary in size and absence of nutrients. Gene expression in prokary-
from a few hundred DNA bases otes occurs primarily at the level of transcription
to more than 2 million bases.
The Human Genome Project has
estimated that humans have be- Gene Regulation in Prokaryotes :
tween 20,000 and 25,000 genes.
In bacteria the expression teins are of two types.
Every person has two cop- of genes is controlled by They are positive regula-
ies of each gene, one inherited
extracellular signals often tors called activators and
from each parent. Most genes
present in the medium in negative regulators called
are the same in all people, but a
small number of genes (less than which bacteria are grown. repressors. These activators
1 percent of the total) are slight- These signals are carried to and repressors are DNA
ly different between people. Al- the genes by regulatory binding proteins
leles are forms of the same gene proteins. Regulatory pro-
with small differences in their
sequence of DNA bases. These
small differences contribute to Negative Regulators or Repressors:
each person’s unique physical
The repressor or inhibitor protein binds to the target site
features.
(operator) on DNA. These block the RNA polymerase en-
zyme from binding to the promoter, thus preventing the tran-
scription. The repressor binds to the site where it overlaps the
polymerase enzyme. Thus, activity of the genes is turned off.
It is called negative control mechanism.
An anti-repressor or anti-inhibitor called inducer is needed to inac-
tivate the repressor and thereby activating the genes. Thus, the
genes are switched on. This is demonstrated by lactose operon.

By : Naseer Ahmed
 What are OPERONS?
bacteria genes that en- expression. The oper- Repressible operons:
code for proteins with on can therefore be
 The include genes
closely related functions categorized as induci-
that encode for en-
are found grouped along ble or repressible.
zymes involved in
with
This cis-acting
story can fitregulatory
100-150
Inducible operons: biosynthetic path-
elements
words. that determine
ways, and the expres-
the transcription of these  They include
The subject matter that ap- sion of the gene is
genes, thus these genes genes that encode
pears in newsletters is virtu- controlled by the end
are regulated in a coordi- for enzymes that -product of the path-
nated way. These
ally endless. clusters
You can include
take part in meta- way
of genes are called oper-
stories that focus on current
bolic pathways the
ons, and their
technologies transcrip-
or innovations expression of the  Example: the "Trp
tion product
in your field. is a single gene is controlled Operon".
polycistronic mRNA. by the substrate
You may also want
Organization to notein
of genes
business orcontributes
economic trends,  Example: the "Lac
operons to Operon"
the regulation of gene
or make predictions for your

What are OPERATORS?

These are segments of DNA that regulate the activity of the structural genes of the
operon.
 If the operator is not bound by a repressor molecule, then the RNA polymer-
ase can pass over the operator and move to the protein coding genes
 If the operator, on the other hand, is bound by a repressor molecule, then the
RNA polymerase is blocked behind the repressor molecule.
 However, when an "Inducer molecule" is present, it binds the repressor mole-
cule causing it to change shape, rendering it incapable of binding the operator.
The RNA polymerase moves freely
 The lactose operon:
 The lactose operon contains 3 genes, Lac Z, Y and A. These genes encode for enzymes required to
metabolize lactose --> beta-galactosidase, lactose permease, and beta-galactoside transacetylase.
 Another regulatory gene, Lac I, is expressed separately and lies upstream of the operon. This gene
ecodes for the lac repressor which regulates the expression of Lac Z, Y ans A
In the absence of lactose:
1. The lac repressor bind to a DNA sequence called the "operator" (found between the lac Z gene
and the lac promoter)
2. In this way the lac repressor blocks the path of RNA polymerase to reach the lac Z,Y and A
genes --> operon remains switched off.

In the presence of lactose:

1. Lactose molecules are metabolized by the lac enzymes, an intermediate is formed called allolac-
tose (an isomer of lactose)
2. Allolactose acts as an inducer by binding to the lactose repressor and changing its conformation -
-> therefore it can no longer bind to the operator.

 In the presence of glucose (even if present together with lactose):Glucose is preferred because it
requires less energy expenditure to be broken down
 The operon senses the glucose presence and by a mechanism called "catabolite repression" it is
switched off.
 The regulatory protein "CAP" (Catabolite Activator Protein) binds to a DNA sequence upstream
to the lac promoter and enhances binding of the RNA polymerase leading to an increased tran-
scription of the operon.
 However, CAP will only bind behind the promoter only if cAMP is bound on it. Adenylate
cyclase though, the enzyme required for the synthesis of cAMP, is inhibited by glucose and the
operon will be eventually expressed at very low rate.

The Tryptophan operon (Trp operon):

1. It includes 5 genes involved in Tryptophan synthesis

2. The genes are expressed as a single mRNA strand, transcribed from an upstream
promoter
3. Another regulatory gene encodes for a trp repressor.
4. There is also the trp operator that is found just downstream from the promoter
5. If tryptophan is present it will bind the trp repressor. This enables it to bind the oper-
ator and block the RNA polymerase. This is a model of end-product inhibition, since
tryptophan is the end product of tryptophan biosynthesis.
Attenuation
 Trp operon attenuation
 Alternatively another method of sup-
pressing gene expression is "attenuation".
This is only possible in prokaryotes since
t has to do with the fact that the mRNA is
translated as soon as it is being tran-
scribed.
 The trp mRNA can form 2 stem-looped
structures between the trp-promoter and
the 1st trp gene. The large stem loop does
not influence transcription and occurs
upstream of the smaller stem loop which
acts as a transcription terminator. If the
large stem loop is not present then the
short stem-loop will be formed.
 Just prior to the stem-loop region fo the
mRNA, there is an open reading frame of
14 codons, of which 2 of them code for
tryptophan.

 The formation of the stem-loops on the mRNA depends on the binding of ribosomes to the mRNA.
 If the tryptophan is present in adequate levels in the cytoplasm, it will be translated and the ribosome will
keep translocating following closely behind the RNA polymerase preventing the formation of the large loop
structure, allowing the terminator structure to form further on and transcription ends.
 If tryptophan is lacking, the ribosome will be stalled as it tries to translate the coding region. This will allow
the RNA polymerase to move ahead and leave enough space for the larger stem-loop structure to form, thus
allowing the transcription to continue.
 Overall the trp repressor determines whether to switch on or off the entire operon while the method of attenu-
ation determines the efficiency of transcription, according to the level of tryptophan available.

Positive Control of Transcription:

The system of regulation in lactose and tryptophan operon is essentially a negative control in the sense that the
operon is normally “on” but is kept “off’ by the regulator protein. In other words, the structural genes are not
allowed to express unless required.

Catabolic Repression:
Lac operon also shows positive control by catabolic repression. This is an additional control system, which binds
the repressor-operator. In E. coli, in the presence of both glucose and lactose, the glucose in first fully utilized and then lac-
tose is taken up for production of energy.Glucose is richest and more efficient source of energy. Glucose has an inhibitory
effect on the expression of lac operon. The mechanism of positive control enables E. coli to adapt more efficiently to the
changing environment of its natural habitat, which is the human intestine.
In the presence of glucose, synthesis of β-galactosidase enzyme becomes suppressed. The inhibitory effect of glu-
cose is due to the marked drop in the level of a nucleotide called cyclic AMP (c-AMP), which inhibits the transcription of
mRNA. Lactose operon transcription requires not only cyclic AMP but also another protein called catabolic activator protein
(CAP). The cAMP and CAP form a complex called cAMP-CRP complex, which is necessary for the functioning of lactose
operon.
A catabolic breakdown product of glucose, called glucose catabolite, prevents the activation of lac operon by lac-
tose. This effect is called catabolic repression. When glucose concentration increases, the cAMP concentration decreases and
vice versa. High concentration of cAMP is necessary for the activation of lac operon.
Normally in the presence of glucose, the lactose operon remains inactive.Glucose catabolite prevents the formation cAMP-
CRP complex. In this way cAMP-CRP system is positive control because expression of lac operon requires the presence of
an activating signal which is this case in cAMP-CRP complex. There are some promoters on DNA at which RNA polymer-
ase cannot initiate transcription without the presence of some additional protein factors such as cAMP-CRP complex. These
factors are positive regulators because their presence is necessary to switch on the cistrons. These are called activators or
stimulators.