2015 Biochemistry of Nucleic Acids

Topic: Activity of Gene

Regulation in Prokaryotes

Submitted to: Dr. Javed Iqbal

Submitted by: Jannat Iftikhar

B11-16

7th semester

Department of Botany

University of the Punjab

Lahore.

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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

Ribosomal Proteins Are Translational Repressors of Their Own

Synthesis

8. Conclusion

9. References

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“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

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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

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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

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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)

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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

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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)

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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

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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)

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Fig.5. lac operon; system turned off in the absence of lactose, and turned on in the presence of

lactose

Positive control by 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

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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

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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

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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.

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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

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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)

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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

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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.

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9. References Essentials of molecular biology, George M. Malancinski, 4 th

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