promoters in gene expression

Role of Promoters in gene expression Ms.Smita

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PROMOTERS:

A promoter is a DNA sequence that can recruit transcriptional machinery

and lead to transcription of the downstream DNA sequence. The specific

sequence of the promoter determines the strength of the promoter (a

strong promoter leads to a high rate of transcription initiation).

In addition to sequences that "promote" transcription, a promoter

may include additional sequences known as operators that control the

strength of the promoter.

For example, a promoter may include a binding site for a protein that

attracts or obstructs the RNAP binding to the promoter. The presence or

absence of the protein will affect the strength of the promoter. Such a

promoter is known as a regulated promoter.

An input/output description of promoter function

Sometimes, we ignore the details of how a promoter works and think of a

promoter as a device that converts inputs into outputs. You can do this

when designing a multi-component system that includes promoters whose

activity must be regulated by other species in the system. A promoter can

be thought of as a device that outputs a certain number of transcribing RNA

polymerases per unit time. Promoters can have different numbers of

inputs. A constitutive promoter has no inputs. Technically, even a

constitutive promoter has inputs, such as the level of free RNA polymerase,

but we often assume that levels of free RNA polymerase are either

unchanging, or never be the limiting factor in transcription initiation. The

level of a repressor that negatively regulates a promoter is an input to a

promoter.

Types of promoters used to regulate gene expression

Promoters used in biotechnology are of different types according to the

intended type of control of gene expression. They can be generally divided

into:

1. Constitutive promoters: These promoters direct expression in virtually

all tissues and are largely, if not entirely, independent of environmental

and developmental factors. As their expression is normally not conditioned

by endogenous factors, constitutive promoters are usually active across

species and even across kingdoms.


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2. Tissue-specific or development-stage-specific promoters: These

direct the expression of a gene in specific tissue(s) or at certain stages of

development. For plants, promoter elements that are expressed or affect

the expression of genes in the vascular system, photosynthetic tissues,

tubers, roots and other vegetative organs, or seeds and other reproductive

organs can be found in heterologous systems (e.g. distantly related species

or even other kingdoms) but the most specificity is generally achieved with

homologous promoters (i.e. from the same species, genus or family). This

is probably because the coordinate expression of transcription factors is

necessary for regulation of the promoter's activity.

3. Inducible promoters: Their performance is not conditioned to

endogenous factors but to environmental conditions and external stimuli

that can be artificially controlled. Within this group, there are promoters

modulated by abiotic factors such as light, oxygen levels, heat, cold and

wounding. Since some of these factors are difficult to control outside an

experimental setting, promoters that respond to chemical compounds, not

found naturally in the organism of interest, are of particular interest. Along

those lines, promoters that respond to antibiotics, copper, alcohol, steroids,

and herbicides, among other compounds, have been adapted and refined to

allow the induction of gene activity at will and independently of other

biotic or abiotic factors.

4. Synthetic promoters: Promoters made by bringing together the

primary elements of a promoter region from diverse origins.

4 Different Plant Promoters in Gene Construct

Some of the plant promoters in gene construct are as follows:

1. Constitutive Promoters 2. Tissue-Specific Promoters 3.Inducible

Promoters 4. Synthetic Promoters.

As a rule, promoters of all structural genes that encode proteins are located

upstream of the start site, the site from which transcription begins. The

promoter determines (1) the level of expression, (2) the developmental


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stage and/or the tissues in which it will be expressed, and (3) the

physical/chemical factor by which the gene expression will be regulated.

A promoter suitable for gene expression in prokaryotes will not function in

eukaryotes, and vice-versa. Further, animal promoters are not suitable for

plants, and promoters that function well in dicots are usually much less

active in monocots, and vice-versa. Therefore, considerable thought has to

be given to the selection of an appropriate promoter for transgene

expression.

A variety of promoters are used to drive transgenes in plants, some of

which are listed in Table below.

These are essentially naturally occurring promoters belonging to the

following three broad groups: (1) constitutive promoters, (2) tissue-

specific promoters, and (3) promoters activated by specific

physical/chemical factors. In addition, number of (4) synthetic promoters

have been developed to achieve a defined regulation of the transgene

expression.


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Table: Some of the constitutive promoters used for driving the expression of

transgenes in different plant species.

1. Constitutive Promoters:

Genes driven by constitutive promoters are expressed in all the tissues and

during all developmental stages of the organism, and their expression is

largely unaffected by physical chemical stimuli. Some examples of such

promoters are 35S promoter, ubiquitin (Ubi) promoter, actinl (Act1)

promoter, nopaline synthase (nos) promoter, octopine synthase (ocs)

promoter, mannopine synthase (mas) promoter, etc.

The 35S, nos ocs, and mas promoters have been obtained from plant

pathogens, and were the first to be used; Ubil and Actl promoters are from

plant genes. In case of many constitutive promoters, the level of gene

expression in different tissues may show some variation, and some

promoters may respond to physical/chemical stimuli.

For example, Actl promoter contains elements that appear to negatively

regulate promoter activity in a tissue- specific manner, particularly in

roots. Similarly, Ubi 1 promoter shows some increase in promoter activity

in response to temperature stress.

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Use of constitutive promoters to produce transgenic plants offers

certain advantages:

(1) They can be used to drive scorable/selectable reporter genes, which are

critical for molecular biology studies, and for the development of

transgenic plants.

(2) Their use will be essential in the case of such proteins that are required

in all tissues and/or during all stages of plant development. Finally,

(3) They will be useful in driving genes encoding such transcription factors

that are involved in transcription regulation.

The 35S promoter is the most commonly used constitutive promoter in

dicot plants, but it does not work satisfactorily in most monocot species.

This promoter is ideal for driving marker genes. Some modifications of 35S

promoter show several-fold increase in their activity.

Maize ubiquitin gene (Ubil) promoter includes the first intron of this gene.

It is reported to function well both in monocots and dicots, and its activity

increases transiently in response to temperature stress. The promoter of

rice actin gene (Actl) shows strong constitutive activity in monocots. As in

the case of Ubil, the presence of first intron of the gene Actl is critical for

efficient function of the Actl promoter.

Summary

There are several advantages to using constitutive promoters in expression

vectors used in plant biotechnology, such as: •High level of production of proteins used to select transgenic cells or

plants;


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•High level of expression of reporter proteins or scorable markers, allowing easy detection and quantification; •High level of production of a transcription factor that is part of a regulatory transcription system; •Production of compounds that requires ubiquitous activity in the plant;

and •Production of compounds that are required during all stages of plant

development.

The first constitutive promoters used for the expression of transgenes in

plants were isolated from plant pathogens.

The search for other constitutive promoters has continued, especially to

identify control regions that are able to drive expression of transgenes in

monocots. In some monocots such as cereals, it has been found that

sequences present in 5' untranslated transcribed regions (e.g., introns) of

certain structural genes are essential for efficient gene expression. Thus,

promoters that work well in dicots, which lack introns, do not generally

work well in monocots.

2. Tissue-Specific Promoters:

Tissue-specific or organ-specific promoters enable the expression of

concerned genes in specific tissues/organs or during certain stages of

development. These promoters drive those genes, which are expressed

specifically in roots, tubers, vascular bundles, seeds, etc. (Refer Table

below)

For example, vicilin and PHA promoters are seed-specific promoters,

TA29 is a tapetum-specific promoter, and Bcpl is specific to both

tapetum and microspores. Although heterologous promoters, i.e.,

promoters from other species, can be used, it is preferable to use

homologous promoters, i.e., promoters from the same species.

Tissue-specific promoters are indispensable in such cases where it is

desired to limit the expression of transgene to a specific


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tissue/organ/developmental stage. For example, promoter TA29 has been

used to drive barnase gene in order to produce male sterility.

This gene encodes an RNAse, which causes cytotoxicity to the tissue in

which it is expressed. Since TA29 promoter is tapetum- specific, it limits

expression of barnase to tapetum cells leading to male sterility without any

adverse effect on other tissues of the transgenic plants.

Similarly, seed-specific promoters limit the expression of transgenes to

seed, and are ideal for driving genes encoding seed storage proteins.

Tissue-specific expression of transgenes would reduce the cost of

transgene expression to the plant. It would also reduce selection pressure

against the insect pest (in cases of trangenes confering insect resistance)

and, thereby, reduce the risk of development of resistance by the insect

pest.

Summary

As mentioned in the Introduction, there are promoters controlling gene

expression in a tissue-dependent manner and according to the

developmental stage of the plant. The transgenes driven by these type of

promoters will only be expressed in tissues where the transgene product is

desired, leaving the rest of the tissues in the plant unmodified by transgene

expression.

Tissue-specific promoters may be induced by endogenous or exogenous

factors, so they can be classified as inducible promoters as well.

Unlike constitutive expression of genes, tissue-specific expression is the

result of several interacting levels of gene regulation. As such, it is then

preferable to use promoters from homologous or closely related plant

species to achieve efficient and reliable expression of transgenes in

particular tissues.


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The purpose of this section is to present those patents directed to plant

tissue-specific promoters in broad terms: •Root promoters: Root promoters that enhance or suppress the

expression of a linked gene in root cells. In addition, the invention

comprises methods for the identification and isolation of plant tissue-

specific promoters in general. •Fruit promoters: A tissue-specific promoter includes fruit specific

promoters that control the expression of genes in mature ovary tissue of a

fruit and in the receptacle tissue of accessory fruits such as strawberry,

apple and pear. The genes driven by the promoters influence fruit

development and ripening. •Seed promoters: University of California have granted patents and patent

applications drawn to seed-specific promoters in broad terms.

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Transcription cassettes having a seed-specific promoter and recombinant

molecules containing a seed-maturation promoter are part of the

inventions.

3. Inducible Promoters:

Promoters that are activated in response to a specific physical factor,

e.g., light, temperature, heat, cold, wound etc., or a specific chemical

compound are called inducible promoters or, sometimes, stimulus-

responsive promoters. These promoters, especially chemical- inducible

promoters, provide fine control on the regulation of gene expression.

A number of native plant gene promoters are stimulus responsive, e.g.,

Adhl promoter (responds to anaerobic condition), cab promoter and

rbcs promoter (respond to light), etc. (Refer above table). But chemical

inducible promoters are synthesized from promoter sequences of different

organisms.

It is important that chemically-regulated promoters should be derived from

such organisms that are as distantly related to plants as possible, e.g., from

bacteria like E. coli, yeast, Drosophila, and mammals.

A tetracycline- regulated system that can either activate (negative

regulation) or repress (positive regulation) transcription in the presence of

tetracycline has been developed.

Some examples of environment-responsive promoters are those of the

genes rbcs, Adhl, cab, hsp, etc. Nuclear gene rbcs encodes the smaller

subunit of RuBISCO (ribulose-1, 5-bisphosphate

carboxylase/oxygenase). This gene is expressed mainly in mature leaves

and, to a lesser extent, in stem and young leaf tissues, and its expression is

induced by light.

The – 166 to – 149 bp sequence of rbcS gene functions as light response

element (LRE) and is responsible for the expression of rbcS gene in

response to light. The alcohol dehydrogenase 1 (Adhl) gene of maize is

expressed only under anaerobic conditions due to the action of a

silencer sequence that suppresses expression in the presence of

oxygen.


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A number chemically regulated gene expression systems have been

constructed using genes and operator systems, preferably, from unrelated

organisms. Tetracycline-regulated system was the first such system to be

developed. Subsequently, several such expression systems were devised,

e.g., metal- regulated, steroid-regulated, plant hormone-regulated,

pathogenesis-regulated, wound-regulated, etc. expression systems.

Summary

As their name says, the activity of these promoters is induced by the

presence or absence of biotic or abiotic factors. Inducible promoters are a

very powerful tool in genetic engineering because the expression of genes

linked to them can be turned on or off at certain stages of development of

an organism or in a particular tissue.

This section presents a general view of promoters whose activity is

triggered by either chemical or physical factors. There are virtually

hundreds of inducible promoters that vary according to the organism

source and cells or tissues where they regulate gene transcription.

Inducible promoters are grouped as: •Chemically-regulated promoters, including promoters whose

transcriptional activity is regulated by the presence or absence of

alcohol, tetracycline, steroids, metal and other compounds. •Physically-regulated promoters, including promoters whose

transcriptional activity is regulated by the presence or absence of

light and low or high temperatures.

Chemically-regulated promoters

The activity of this class of promoters is modulated by chemical compounds

that either turn off or turn on gene transcription. As prerequisites, the

chemicals influencing promoter activity typically: • should not be naturally present in the organism where expression of the

transgene is sought;


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• should not be toxic; • should affect only the expression of the gene of interest; • should be easy to apply or removal; and • should induce a clearly detectable expression pattern of either high or very low gene expression for their optimal use as modulators of gene

expression.

Physically-regulated promoters

Promoters induced by environmental factors such as water or salt stress,

anaerobic condition, temperature, illumination and wounding have

potential for use in the development of plants resistant to various stress

conditions. These promoters contain regulatory elements that respond to

such environmental stimuli.

Temperature-induced promoters include cold- and heat-shock-induced

promoters. In many cases, these promoters are able to operate under

normal temperature conditions, which vary according to the organism, but

when either cold or heat is applied, the promoters maintain activity. In

addition, expression can be enhanced by the application of higher or lower

temperature as compared to the normal temperature conditions. One of the

best studied eukaryotic heat-shock systems is the one found in

Drosophila (fruit fly).

4. Synthetic Promoters:

A promoter assembled by combining various primary elements required

for the defined promoter function may be referred to as a synthetic

promoter. The various elements used in such a promoter are, usually, of

diverse origin. Among these elements the TATA box, the transcription start

site or cap site, and the CCAAT consensus sequence are required for

accurate constitutive transcription. A synthetic promoter may be either

constitutive tissue-specific or inducible, depending on the functional

sequences used in its construction.

Attempts have been made to increase the activity of 35S promoter by

adding specific modules to the native promoter. Inclusion of an additional


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copy of the 35S enhancer (-208 to -46 bp), i.e., double 35S enhancer, caused

a 6-fold increase in 35S promoter activity.

Summary

As mentioned in the introductory information about promoters, a set of

minimum elements are required for an activity of eukaryotic promoter.

Among those elements are the TATA box, the transcription start site or

CAP site and the CCAAT consensus sequence, which is required for

accurate transcription.

From the sequences of these elements in diverse organisms, it is possible to

synthesize consensus sequences that may work across different organisms

and are not necessarily derived from a particular organism.

The group of patents under this section are directed to promoters whose

parts are synthesized as consensus sequences of the promoter elements

found in nature.

promoters in gene expression

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