Plant Promoters

What are Cis & Trans acting elements/

factors ?

Cis-acting elements - DNA sequences in the vicinity
of the structural portion of a gene that are required
for gene expression

Trans-acting factors - Proteins that bind to the cis-
acting sequences to control gene expression

The expression of a particular gene may be
regulated by the concerted action of both cis and
trans-acting elements.
Promoters:
The key cis-acting regulatory region that
controls the transcription of adjacent
coding region(s) into mRNA.
It acts as the regulator for the level of gene
expression.
Different promoters can give genes
different expression patterns.
In plants the 5' flanking region contains
Core promoter
Regulatory elements
Upstream enhancer sequences

Promoter and Enhancer
Source: Genes VIII Benjamin Lewin

Core promoters:
Core promoters are Minimal promoters that contain TATA
box.
It is also called Goldberg-Hogness box.
Essential for RNA pol -II dependent transcription
Located at about -20 to -35 bp upstream from
transcription initiation sites (TIS).
Regulates the binding of transcription factor -TFIID to
TIS.

Eukaryotic Promoters
CAAT box.
A consensus sequence close to -80 bp from the start point (+1).
It plays an important role in promoter efficiency, by increasing its
strength, and it seems to function in either orientation.
This box is replaced in plants by the AGGA box
TATA box.
located around - 25 bp upstream of the start point.
The TATA box tends to be surrounded by GC rich sequences.
The TATA box binds RNA polymerase II and a series of transcription
factors to form an initiation complex.
GC box.
Found in multiple copies in the promoter region- surrounding the
TATA box
CAP site.
A transcription initiation sequence or start point defined as +1, at
which the transcription process actually starts
Eukaryotic Promoters
Conserved eukaryotic promoter
elements
Consensus sequence
CAAT box GGCCAATCT
TATA box TATAA
GC box GGGCGG
CAP site TAC
Eukaryotic Promoters
Types of Promoters
Constitutive promoters:
Inducible promoters:
Tissue-specific promoters :
Synthetic promoters:
Constitutive promoters
Constitutive Promoters
Facilitate expression of the gene in all tissues regardless of the
surrounding environment and development stage of the
organism
They are active in all circumstances in the cell.
Examples:


Plant pathogen/
Dicot promoters
Monocot promoters

Opine promoters
CaMV 35S promoter

Plant ubiquitin promoter (Ubi)
Rice actin 1 promoter (Act-1)
Maize alcohol dehydrogenase 1
promoter (Adh-1)
CaMV 35S Promoter
Cauliflower mosaic virus (CaMV) replicate through reverse
transcription just like retroviruses.
The promoter of the 35S RNA is a very strong constitutive
promoter responsible for the transcription of the whole
CaMV genome.
Most widely used promoter in plant gene transfer.
Drives high level of expression in dicots
Its ideal for driving expression of selectable marker genes
and in some cases of reporter genes.

Inducible Promoters:
Inducible promoters
They may be Chemically or Physically induced

Types & Example:

Chemically induced
1. Alcohol-regulated:
2. Tetracycline-regulated:
3. Steroid-regulated:
4. Metal-regulated:
5. Pathogenesis-related

Physically induced
1. Temperature-regulated promoters
2. Light-regulated promoters


Inducible Promoters:
The activity of these promoters is induced by
the presence of biotic or abiotic factors.
The expression of genes operably linked to
them can be Turned On or Off at certain
stages of development of an organism or in a
particular tissue.
Desirable features of a inducible
promoters:
1. No expression of Trans Gene in the
absence of inducer
2. It should be specific to inducer- to only
one or one class of inducer.
3. The inducer should be non toxic
4. Induction of gene expression should be
rapid following the application of inducer
5. Gene expression should cease rapidly
following the withdrawal of inducer


Types of inducible promoters:
Three types:
1. Non plant derived systems
2. Plant derived systems that respond to
environmental signals
3. Plant derived systems based on
developmental control of gene expression.
1. Non plant derived systems
A. Tetracycline
B. Ethanol Inducible
C. Steroid Inducible
D. Copper Inducible
Tetracycline
Tetracycline is used to either De- Repress Or Inactivate Gene Expression
De- repression:
Tetracycline repressor (TetR) binds to tet operator- negative regulation
Tetracycline act as a inducer- activation / de repression.
Inactivation:
TetR is modified to an activator called Tetracycline Trans-activator (tTA).
tTA protein is created by fusing TetR (Escherichia coli), with the activation
domain of another protein, VP16 (Herpes Simplex Virus)
tTA binds to operators and induces gene expression in absence of
tetracycline
When Tetracycline is present , tTA is released and expression ceases.
Tetracycline-controlled
transcriptional activation
P
tet
promoter expresses
TetR - the repressor
TetA- the protein that
pumps tetracycline
antibiotic out of the
cell
Derepression by
doxycycline ( Dox) /
Tetracycline
Source: http://en.wikipedia.org/wiki/Tetracycline-controlled_transcriptional_activation
Alcohol inducible:
Source: Aspergillus nidulans
a well-characterized positive operon system- controls the cellular
response to ethanol.
The first enzyme in the pathway is Alcohol Dehydrogenase I
encoded by the alcA gene.
The transcriptional activator protein AlcR binds target sequences
within the alcA gene promoter in the presence of ethanol, ethyl
methyl ketone or other alcohols/ketones.
These compounds act as inducers of the gene expression.
http://www.patentlens.net/daisy/promoters/271/272/g1/280.html
Alcohol inducer:
Promoter system transferred into plants:
One system transferred into plants provides
alcR gene under the control of a strong constitutive promtoer
such as CaMV 35S
Modfied alcA promoter linked to a gene of interest.
Ethanol act as a inducer-AlcR binds to promoters and activates
transcription
Syngenta has several patents and patent applications in Europe and Australia directed to the
transcriptional system containing the alcohol dehydrogenase I (alcA) gene promoter
http://www.patentlens.net/daisy/promoters/271/272/g1/280.html
http://www.patentlens.net/daisy/promoters/271/272/g1/1356.html
Steroid Inducible
Modified transcription factor capable of binding with steroid
inducer acts as activator of chimeric promoters
The mammalian Glucocorticoid Receptor (GR) is a member of the
family of animal steroid hormone receptors.
GR acts as a transcription factor by activating gene transcription
from promoters containing Glucocorticoid Response Elements
(GRE)
A system comprised of the GR and GREs resulted in transient
expression in tobacco
Dexamethasone ( a strong synthetic glucocorticoid ) used as
inducer.
Oestrogen and ecdysone are other inducers
Steroid Inducible
Ecdysone based Systems:
Ecdysone is a steroidal prohormone of the
major insect molting hormone - secreted from
the prothoracic glands
It also respond to non steroid ecdysone agonist
RH5992 ( Tebufenozide)
Acicada moulting
Copper Inducible
This system is based on Metallothionein genes from
yeast- ACE1
Upon binding Copper, the transcription factor binds to
Chimeric Promoter and activates the expression.
Patents:
Genentech has a granted US patent related to inducible
transcription control sequences isolated from a yeast
metallothionein gene.
2. Plant derived systems that
respond to environmental signals
Wound inducible
Heat shock inducible
Wound inducible
The nos promoter (nopaline synthase) is wound inducible in
both vegetative and reproductive organs.
The induction of the nos promoter was observed in leaves,
stems, cotyledons, and various reproductive organs.
The response is not organ specific.
The wound response was further enhanced by addition of
auxins.
Nopaline synthase promoter is wound inducible and auxin inducible. G An, M
A Costa and S B Ha (http://www.plantcell.org/content/2/3/225.abstract)
Wound inducible
The promoter, found in potato plants, regulates a gene
encoding the serine-type proteinase inhibitor II (PinII).
PinII is constitutively expressed only in tubers and young
floral buds of potatoes
PinII expression can be induced by mechanical wounding
in leaves
Methyl- jasmonate mimic wounding-as inducer
Used for expression of pest resistant genes
http://myip.cctec.cornell.edu/techdocs/D1155.D1155_Wound_Inducible_Promoter_Tech_Brief.pdf
Heat Shock Inducible:
Promoter regions of HS gene contain HSE- Heat
Shock Response Elements.
HSE mediate the heat shock inducibility of genes.
Contain unique palindrome motif of nGnnTTc
and nnGAA

Heat shock inducible
HEAT SHOCK ELEMENT(HSE) MEDIATED HEAT SHOCK INDUCIBILITY
Soybean heat-shock promoter Gmhsp17.3
Temp. shock of - 40
0
C produced 10k to 20k nbr of copies of heat shock protein
The promoter of the HSP18.2 gene from Arabidopsis, encoding a heat
shock protein, indicated that it functions as a strong inducible system in
plants
1. Comparison of different constitutive and inducible promoters for the overexpression
of transgenes in Arabidopsis thaliana. Holtorf S, Apel K, Bohlmann H.(
http://www.ncbi.nlm.nih.gov/pubmed/8541491)
2. Gene silencing using a heat-inducible RNAi system in Arabidopsis (http://hal.archives-
ouvertes.fr/docs/00/11/95/62/PDF/BBRC_HAL.pdf )
3. Plant derived systems based on
developmental stage:
Senescence Specific Gene Expression
ABA Inducible Gene Expression
Auxin Inducible Gene Expression
Senescence Specific Gene Expression
Senescence inducible promoters from Arabidopsis genes-
SAG12 & SAG13( Senescence Associated Gene)
An ipt gene(isopentenyltransferase) under
control of the senescence specific SAG12 promoter
significantly delayed developmental and postharvest leaf
senescence in mature heads of transgenic tobacco .
Similar result in In lettuce
ABA Inducible Gene Expression
Abscisic Acid (ABA) can be used as a highly effective
chemical inducer
ABA controls expression of large nbr of genes.
ABA is mediates a wide variety of stress responses in
plants
ABA induction require two fragment s of promoters
ABRE (ABA Response Element)
CE ( Coupling Element)


Auxin Inducible Gene Expression
Auxin plays an essential role in :
Shoot and lateral root formation
Apical dominance
Tropism
Senescence
A large number of auxin-inducible genes have been cloned and
characterized, including Arabidopsis, soybean hypocotyls, pea epicotyls etc
Examples:
1. The auxin-inducible soybean GH3 gene
2. ARGOS, a novel auxin- inducible gene that is involved in organ size
control in Arabidopsis
AuxREs( auxin response elements) combined with 35S core promoter

Tissue Specific Promoters
Promoters which operate in particular tissues and at certain
developmental stages of a plant.
They may be induced by endogenous and exogenous factors.
Root Promoters ,Fruit Promoters ,Seed Promoters etc.
Example:
Seed storage proteins:
legumin box : 5-TCCATAGCCATGCAAGCTGCA-3
Phosphoenolpyruvate (PEP) Carboxylase Promoter which induces
gene expression only in cells that are actively involved in
photosynthesis


Synthetic Promoters:
Promoters made by bringing together the primary
elements of a promoter region from diverse
origins.

Chimeric genes form through the combination of
portions of one or more coding sequences to
produce new genes

By fusing the promoters of two different genes into
one chimeric gene.
Reference:
1. Plant Biotechnology-The genetic manipulation of plants
by Adrian Slater and others
2. Plant Molecular Biotechnology by S. Mahesh
3. Introduction to Plant Biotechnology by H. S. Chawla
4. Cellular And Biochemical Science edited by G. Tripathi