gene regulation in prokaryotes

1. 2015
Biochemistry of Nucleic Acids
Topic: Activity of Gene
Regulation in Prokaryotes
Submitted to: Dr. JavedIqbal
Submitted by: Jannat Iftikhar
B11-16
7th
semester
Department of Botany
University of the Punjab
Lahore.

2. 1
Contents
1. Introduction
2. Discovery
3. Gene regulation in prokaryotes
4. Regulation of transcription
5. Principles of Transcriptional Regulation
6. Lac operon
 Structure of lac operon
 Mechanism
 Positive control by CAP-cAMP complex
7. Tryptophan operon
 Structure of trp operon
 Mechanism
 Attenuation
 RibosomalProteins Are Translational Repressors of Their Own
Synthesis
8. Conclusion
9. References

3. 2
“Activity of Gene Regulation in
prokaryotes”
1. Introduction:
Gene is the segment of DNA that controls all traits of organism that may be
physical or metabolical. In information encoded in DNA is transcribed into RNA
and then translate into proteins. The ability of cell to switch the genes on and off
is of fundamental importance because it enables the cell to respond to the
changing environment and it is the basis of cell differentiation. So we can say
that:
“Gene Regulation is a process in which a cell determines which genes it
will express and when.”
Regulation of gene expression includes a wide range of mechanisms that are
used by the cell to increase or decrease the production of specific gene products
(protein or RNA). Sophisticated programs of gene expression are widely
observed in biology to trigger developmental pathways, respond to environmental
stimuli or adapt to new food sources.
2. Discovery:
Gene regulation is essential for viruses, prokaryotes and eukaryotes as it
increases the versatility and adaptability of an organism by allowing the cell to
express protein when needed. Although as early as 1951 Barbara
McClintock showed interaction between two genetic loci, Activator (Ac) and
Dissociator (Ds), in the color formation of maize seeds, the first discovery of a
gene regulation system is widely considered to be the identification in 1961 of
the lac operon, discovered by Jacques Monod, in which some enzymes involved
in lactose metabolism are expressed by the genome of E.coli only in the
presence of lactose and absence of glucose. Furthermore, in 1953, Jacques
Monod discovered Enzyme Repression. He observed the presence of
tryptophan in medium of E.coli which repressed the synthesis of tryptophan
synthetase and then further studies showed that all the enzymes, present in
tryptophan biosynthetic pathway are simultaneously repressed in the presence of
end product.
In bacteria such as E.coli, Salmonella and Streptomyces steps controlling genes
in biosynthetic pathway are adjacent on their chromosomes ad they can be
coordinately repressed. In eukaryotes, related genes are present in genome in
scattered form, so coordinate control becomes more difficult to achieve. This

4. 3
cluster of bacterial genes was first observed by Miloslav Demerec and his
colleges in 1956.
3. Gene Regulation in Prokaryotes:
The rate of expression of bacterial gene is controlled mainly at level of
transcription. Regulation can occur at both the initiation and termination of mRNA
synthesis because bacteria obtain their food from the medium that immediately
surrounds them. Their regulation mechanisms are designed to adapt quickly to
the changing environment. If a gene is not transcribed then the gene product and
ultimately the phenotype will not be expressed.
In contrast, eukaryotes have much complex and larger genome and cells of
higher organisms are surrounded by constant internal milieu. The ability of such
cells to respond to harmones and to impulse in nervous system is thus
comparatively more important that the ability to respond rapidly in the presence
of certain nutrients. (fig.1)
Fig.1. Diagram showing at which stages in the DNA-mRNA-protein pathway expression can be
controlled

5. 4
4. Regulation of Transcription:
As in prokaryotes gene regulation occur at transcription level, so Transcription of
a gene by RNA polymerase can be regulated by at least five mechanisms;
 Specificity factors alter the specificity of RNA polymerase for a
given promoter or set of promoters, making it more or less likely to bind to
them (i.e., sigma factors used in prokaryotic transcription).
 Repressors bind to the Operator, coding sequences on the DNA strand
that are close to or overlapping the promoter region, impeding RNA
polymerase's progress along the strand, thus impeding the expression of
the gene. The image to the right demonstrates regulation by a repressor in
the lac operon.
 General transcription factors position RNA polymerase at the start of a
protein-coding sequence and then release the polymerase to transcribe
the mRNA.
 Activators enhance the interaction between RNA polymerase and a
particular promoter, encouraging the expression of the gene. Activators do
this by increasing the attraction of RNA polymerase for the promoter,
through interactions with subunits of the RNA polymerase or indirectly by
changing the structure of the DNA.
 Enhancers are sites on the DNA helix that are bound by activators in
order to loop the DNA bringing a specific promoter to the initiation
complex. Enhancers are much more common in eukaryotes than
prokaryotes, where only a few examples exist.
 Silencers are regions of DNA sequences that, when bound by particular
transcription factors, can silence expression of the gene.
Here, we’ll explain two systems i.e, repression and induction by which
gene regulation is accomplished in prokaryotes.
5. Principles of Transcriptional Regulation:
i. Gene Expression Is Controlled by Regulatory Proteins:
Genes are very often controlled by extracellular signals, in the case of bacteria,
this typically means molecules present in the growth medium. These signals are
communicated to genes by regulatory proteins, which come in two types: positive
regulators, or activators; and negative regulators, or repressors. Typically these
regulators are DNA binding proteins that recognize specific sites at or near the

6. 5
genes they control. An activator increases transcription of the regulated gene;
repressors decrease or eliminate that transcription. First, RNA polymerase binds
to the promoter in a closed complex (in which the DNA strands remain together).
The polymerase-promoter complex then undergoes a transition to an open
complex in which the DNA at the start site of transcription is unwound and the
polymerase is positioned to initiate transcription. This is followed by promoter
escape, or clearance, the step in which polymerase leaves the promoter and
starts transcribing.
ii. Many Promoters Are Regulated by Activators That Help RNA
Polymerase Bind DNA and by Repressors That Block That Binding:
At many promoters, in the absence of regulatory proteins, RNA
polymerase binds only weakly. This is because one or more of the
promoter elements are imperfect. When polymerase does occasionally
bind, however, it spontaneously undergoes a transition to the open
complex and initiates transcription. This gives a low level of constitutive
expression called the basal level. Binding of RNA polymerase is the rate
limiting step in this case. To control expression from such a promoter, a
repressor need only bind to a site overlapping the region bound by
polymerase. In that way repressor blocks polymerase binding to the
promoter, thereby preventing transcription, although it is important to note
that repression can work in other ways as well. The site on DNA where a
repressor binds is called an operator. To activate transcription, an
activator just helps polymerase bind the promoter. Typically this is
achieved as follows: The activator uses one surface to bind to a site on
the DNA near the promoter; with another surface, the activator
simultaneously interacts with polymerase, bringing the enzyme to the
promoter. This mechanism, often called recruitment, is an example of
cooperative binding of proteins to DNA. The interactions between the
activator and polymerase, and between activator and DNA, serve merely
“adhesive” roles: the enzyme is active and the activator simply brings it to
the nearby promoter. Once there, it spontaneously isomerizes to the open
complex and initiates transcription. The lac genes of E. coli are transcribed
from a promoter that is regulated by an activator and a repressor working
in the simple ways. (fig.2)

7. 6
Fig.2. Activation by Recruitment of RNA Polymerase. (a) In the absence of both activator
and repressor, RNA polymerase occasionally binds the promoter spontaneously and
initiates a low level (basal level) of transcription. (b) Binding of the repressor to the operator
sequence blocks binding of RNA polymerase and so inhibits transcription. (c) Recruitment
of RNA polymerase by the activator gives high levels of transcription. RNA polymerase is
shown recruited in the closed complex. It then spontaneously isomerizes to the open
complex and initiates transcription.
iii. Some Activators Work by Allostery and Regulate Steps after RNA
Polymerase Binding:
Not all promoters are limited in the same way. Thus, consider a promoter
at the other extreme from that described above. In this case, RNA
polymerase binds efficiently unaided and forms a stable closed complex.
But that closed complex does not spontaneously undergo transition to the
open complex. At this promoter, an activator must stimulate the transition
from closed to open complex, since that transition is the rate-limiting step.
Activators that stimulate this kind of promoter work by triggering a
conformational change in either RNA polymerase or DNA. That is, they

8. 7
interact with the stable closed complex and induce a conformational
change that causes transition to the open complex. This mechanism is an
example of allostery. Some promoters are inefficient at more than one
step and can be activated by more than one mechanism. Also, repressors
can work in ways other than just blocking the binding of RNA polymerase.
For example, some repressors inhibit transition to the open complex, or
promoter escape. (fig.3)
Fig.3. Allosteric Activation of RNA Polymerase. (a) Binding of RNA polymerase to the
promoter in a stable closed complex. (b) Activator interacts with polymerase to trigger
transition to the open complex and high levels of transcription.
6. The Lac operon:
Lac operon is induced 1000 folds by lactose. The cell is an energy efficient unit
that makes protein it needs. E. coli has about three thousand protein encoding
genes but only a set of them is expressed at any one time.
The best illustration of this efficiency is provided by the enzymes involved in the
utilization of disaccharide lactose. The synthesis of these enzymes may be
induced up to 1000 folds in respond to the addition of lactose to the culture
media. Regulation by enzyme induction has been found in many other bacterial
systems that degrade sugars, amino acids and lipids. In these systems, the
availability of the substrates stimulates the production of enzymes involved in its
degradation. Induction is the production of a specific enzyme (or set of
enzymes) in response to the presence of a substrate. The lac operon is a
inducible system. (fig.4)

9. 8
Fig.4. Diagram representing the lac operon; regulatory gene, structural genes.
 Structure of the lac operon:
 The lac operon consists of the three structural genes, a promoter,
regulator, terminator, regulator and an operator.
 These three structural genes are; lac Z, lac Y and lac A.
 Lac Z encode β-galactosidase, an intracellular enzyme that cleaves the
disaccharide lactose into glucose and galactose.
 Lac Y encode β-galactosidase permease, an inner membrane bound
symporter that pumps lactose into the cell using a proton gradient.
 Lac A encodes β-galactosidase transacetylase, an enzyme that transfer
an ecetyl group from acetyl-CoA to β-galactosides.
 Only lac Z and lac Y are necessary for lactose catabolism.
lac Operon Gene Gene Function
I Gene for repressor protein
P Promoter
O Operator
lac Z Gene for ß-galactosidase
lac Y Gene for ß-galactoside permease
lac A Gene for ß-galactoside transacetylase

10. 9
 Mechanism:
The three enzymes required for the utilization of the lactose are β-galactosidase,
β-galactosidase pemease and β-galactosidase transacetylase. The synthesis of
these enzymes is regulated coordinately as a unit called operon. Operon is the
group of the genes that are nest to each other in DNA and that can be controlled
in a unified manner. The genes of the structural enzymes are always transcribed
together into a single polycistronic lac mRNA which explains why they are always
expressed together.
The expression of lac operon is regulated by lac repressor. A protein having
four identical subunits of 40,000 Dalton each. In an E.coli there are about ten lac
repressor molecules which are coded by the regulatory genes. The repressor
binds closely and specifically to short DNA segment called “operator”, which is
in location very close to the β-galactosidase gene.
The affinity of the repressor to bind to the operator is regulated by inducer,
a small molecule that can bind to the repressor. The natural inducer of lactose is
allolactose, a metabolite of lactose. However, the analogue IPTG (isopropyl
thiogalactosidase) is a more powerful inducer that is preferred in the laboratory.
Each subunit of the repressor has one binding site for inducer and upon binding it
undergoes a conformational change by which it becomes unable to bind to the
operator. In this way the presence of the inducer permits transcription of lac
operon which is no longer block by the reppressor. The effect of this
conformational change is dramatic. While in the absence of lactose, E.coli cells
have an average of only three molecule of β-galactosidase enzyme per cell. After
induction of lac operon here thousands molecules of β-galactosidase are present
in each cell representing 3% of the total proteins.
Promoter is the DNA segment to which RNA polymerase binds when initiating
transcription. Repressor binds within the promoter and prevents attachment
of RNA polymerase. Two sections are particularly important for RNA
transcription, first is TATGTTT sequence located 6-12 bases before the
transcription starting site, which is conserved in most prokaryotic promoters and
second is a region located 35 bases before the beginning of mRNA which is
known to be important because mutation within it will severely inhibit lac
expression. RNA polymerase binds to a region of about 80 nucleotides of DNA
and RNA polymerase binding site, in fact overlaps with the region covered by the
repressor. Studies have shown that repressor bind to the operator blocks the
binding of RNA polymerase. The way in which the repressor work is very simple.
They bind within the promoter and prevents the attachment of RNA polymerase.
(fig.5)

11. 10
Fig.5. lac operon; system turned off in the absence of lactose, and turned on in the presence of
lactose
 Positive controlby CAP-cAMP complex:
cAMP has a regulatory role in bacteria, activating inducible operons at level of
transcription. E.coli has cAMP receptor proteins that is able to bind with cAMP
with high affinity. This protein is also known as catabolite activator protein (CAP).
CAP is a dimeric protein which when complex with the cAMP is able to bind to a
specific site within the lac promoter region. RNA polymerase will recognize the
lac promoter if only the CAP-cAMP complex is already bound to it. Therefore, in
lac operon in addition to the negative control provided by the repressor there is
also a positive control provided by the CAP-cAMP complex.
The positive control mechanism is also required for the expression of many other
inducible proteins, such as those involved in utilization of maltose, galactose and
arabinose. However, cAMP is not necessary for the synthesis of the enzymes
required for the utilization of glucose as an energy source. This mechanism is
highly beneficial in E.coli because its cAMP level varies according to the
available food source. Bacteria growing in the presence of glucose have low
cAMP content than those growing in poorer energy source such as lactose.
When intracellular cAMP level is low i.e, when glucose is available the CAP
protein does not bind to the promoter and lac operon will not turn on even in the
presence of lactose. As a result E.coli grown in the presence of both glucose and

12. 11
lactose, it will use only glucose. This makes good sense because glucose is
richest and most efficient energy source. This clears that this mechanism of
positive control enables the E.coli to adapt more efficiently to changing
environment of natural habitat. (Fig.6)
Fig.6. outline of lac operon, and positive control regulation by CAP-Camp complex

13. 12
7. Tryptophan operon:
In tryptophan operon, regulation of transcription occur at initiation and
termination. In E. coli the five contiguous trp genes encode enzymes that
synthesize the amino acid tryptophan. These genes are expressed efficiently
only when tryptophan is limiting. (fig.7)
 Structure:
Fig.7. Structure of trp operon
Trp operon gene Gene function
trp R Codes for repressor
P promoter
O Operator, site of attachment of RNA polymerase
TrpE Codes for Anthranllate
TrpD Codes for Phosphoribosyl anthranllate transferase
TrpC Codes for Phosphoribosyl anthranllate isomerase
TrpB Codes for Trp β synthatase
trpA Codes for Trp α synthatase

14. 13
 Mechanism:
The genes are controlled by a repressor, just as the lac genes are, but in this
case the ligand that controls the activity of that repressor (tryptophan) acts not as
an inducer but as a co-repressor. That is, when tryptophan is present, it binds the
trp repressor and induces a conformational change in that protein, enabling it to
bind the trp operator and prevent transcription. When the tryptophan
concentration is low, the trp repressor is free of its co-repressor and vacates its
operator, allowing the synthesis of trp mRNA to commence from the adjacent
promoter. Surprisingly, however, once polymerase has initiated a trp mRNA
molecule it does not always complete the full transcript. Indeed, most messages
are terminated prematurely before they include even the first trp gene (trpE),
unless a second and novel device confirms that little tryptophan is available to
the cell.
Fig.8. trp operon; system turned on when tryptophan is absent, system turned off when tryptophan
is present.

15. 14
 Attenuation:
This second mechanism overcomes the premature transcription termination,
called attenuation. When tryptophan levels are high, RNA polymerase that has
initiated transcription pauses at a specific site, and then terminates before getting
to trpE. When tryptophan is limiting, however, that termination does not occur
and polymerase reads through the trp genes. Attenuation, and the way it is
overcome, rely on the close link between transcription and translation in bacteria,
and on the ability of RNA to form alternative structures through intramolecular
base pairing. The key to understanding attenuation came from examining the
sequence of the 5’ end of trp operon mRNA. This analysis revealed that 161
nucleotides of RNA are made from the tryptophan promoter before RNA
polymerase encounters the first codon of trpE. Near the end of the sequence,
and before trpE, is a transcription terminator, composed of a characteristic
hairpin loop in the RNA, followed by eight uridine residues. At this so-called
attenuator, RNA synthesis usually stops (and, we might have thought, should
always stop), yielding a leader RNA 139 nucleotides long. (fig.9)
Fig.9. Trp Operator Leader RNA. Features of the nucleotide sequence of the trp operon leader RNA.
Three features of the leader sequence allow the attenuator to be passed by RNA
polymerase when the cellular concentration of tryptophan is low. First, there is a
second hairpin (besides the terminator hairpin) that can form between regions 1

16. 15
and 2 of the leader. Second, region 2 also is complementary to region 3; thus,
yet another hairpin consisting of regions 2 and 3 can form, and when it does it
prevents the terminator hairpin (3, 4) from forming. Third, the leader RNA codes
for a short leader peptide of 14 amino acids that is preceded by a strong
ribosome binding site. The sequence encoding the leader peptide has a striking
feature of two tryptophan codons in a row. Their importance is underscored by
corresponding sequences found in similar leader peptides of other operons
encoding enzymes that make amino acids. Thus, the leucine operon leader
peptide has four adjacent leucine codons, and the histidine operon leader
peptide has seven histidine codons in a row. In each case these operons are
controlled by attenuation. The function of these codons is to stop a ribosome
attempting to translate the leader peptide; thus, when tryptophan is scarce, little
charged tryptophan tRNA is available, and the ribosome stalls when it reaches
the tryptophan codons. Thus, RNA around the tryptophan codons is within the
ribosome and cannot be part of a hairpin loop. A ribosome caught at the
tryptophan codons masks region 1, leaving region 2 free to pair with region 3;
thus the terminator hairpin (formed by regions 3 and 4) cannot be made, and
RNA polymerase passes the attenuator and moves on into the operon, allowing
Trp enzyme expression. If, on the other hand, there is enough tryptophan (and
therefore enough charged Trp tRNA) for the ribosome to proceed through the
tryptophan codons, the ribosome blocks sequence 2 by the time RNA containing
regions 3 and 4 has been made. Ribosome blocking region 2 allows formation of
the terminator hairpin (from regions 3 and 4), aborting transcription at the end of
the leader RNA. The leader peptide itself has no function and is in fact
immediately destroyed by cellular proteases. The use of both repression and
attenuation to control expression allows a finer tuning of the level of intracellular
tryptophan. It provides a two-stage response to progressively more stringent
tryptophan starvation—the initial response being the cessation of repressor
binding, with greater starvation leading to relaxation of attenuation. But
attenuation alone can provide robust regulation: other amino acid operons like
his and leu have no repressors; instead, they rely entirely on attenuation for their
control. (fig.10)

17. 16
Fig.10. mechanism of transcriptional attenuation of trp operon
 Ribosomal Proteins Are Translational Repressors of Their Own Synthesis:
Regulation of translation often works in a manner analogous to transcriptional
repression: a “repressor” binds to the translation start site and blocks initiation of
that process. In some cases, this binding involves recognition of specific
secondary structures in the mRNA. We consider here the regulation of the genes
that encode ribosomal proteins. Correct expression of ribosomal protein genes
poses an interesting regulatory problem for the cell. Each ribosome contains
some 50 distinct proteins that must be made at the same rate. Furthermore, the
rate at which a cell makes protein, and thus the number of ribosomes it needs, is
tied closely to the cell’s growth rate; a change in growth conditions quickly leads
to an increase or decrease in the rate of synthesis of all ribosomal components.
Control of ribosomal protein genes is simplified by their organization into several
different operons, each containing genes for up to 11 ribosomal proteins. Some
non ribosomal proteins that also are required according to growth rate are
contained in these operons, including RNA polymerase subunits α, β, and β’. As
with other operons, these are sometimes regulated at the level of RNA synthesis.
But, the primary control of ribosomal protein synthesis is at the level of translation
of the mRNA, not transcription. This distinction is shown by a simple experiment.
When extra copies of a ribosomal protein operon are introduced into the cell, the

18. 17
amount of mRNA increases correspondingly, but synthesis of the proteins stays
nearly the same. Thus, the cell compensates for extra mRNA by curtailing its
activity as a template. This happens because ribosomal proteins are repressors
of their own translation. For each operon, one (or a complex of two) of the
ribosomal proteins binds the messenger near the translation initiation sequence
of one of the first genes of the operon, preventing ribosomes from binding and
initiating translation. Repressing translation of the first gene also prevents
expression of some or all of the rest. This strategy is very sensitive. A few
unused molecules of protein L4, for example, will shut down synthesis of that
protein, as well as synthesis of the other ten ribosomal proteins in its operon. In
this way, these proteins are made just at the rate they are needed for assembly
into ribosomes. How one protein can function both as a ribosomal component
and as a regulator of its own translation is shown by comparing the sites where
that protein binds to ribosomal RNA and to its messenger RNA. These sites are
similar both in sequence and in secondary structure. The comparison suggests a
precise mechanism of regulation. Since the binding site in the messenger
includes the initiating AUG, mRNA bound by excess protein S7 cannot attach to
ribosomes to initiate translation. (This is analogous to Lac repressor binding to
the lac promoter and thereby blocking access to RNA polymerase.) Binding is
stronger to ribosomal RNA than to mRNA, so translation is repressed only when
all need for the protein in ribosome assembly is satisfied.
8. Conclusion :
Trp operon and lac operon are two important biosynthetic systems in E.coli that
enable them to adapt quickly according to the changing environment. Any
imbalance or mutation at any stage or in any gene effect their activity and
ultimately their metabolic functions will be disturbed and they will not be able to
cope up with the demands of the environment. Prokaryotes are unicellular or
colonial organisms. A number of genes are present in them but different genes in
the same cell are activated at different times. This also help the cell to spend a
lot of energy to activate different genes that are not required by the cell at
particular time. And in prokaryotes regulation is accomplished at transcription
level.

19. 18
9. References
 Essentials of molecular biology, George M. Malancinski, 4th
edition,2005.
 Genetic structure and function, P.F. Smith Keary, 1975,Halsted
press,New York.
 An introduction to genetic analysis, Grifith AJF,Miller JH, Suzuki DT,
et al, 7th
edition, 2000.
 Genetics by strickberger,3rd
edition.
 http://en.wikipedia.org/wiki/Trp_operon
 http://en.wikipedia.org/wiki/Lac_operon
 http://en.wikipedia.org/wiki/Regulation_of_gene_expression
 biology.kenyon.edu/courses/biol63/watson_16.pdf