Plant Promoters

Promoters:
A site in a DNA molecule at which RNA polymerase and

transcription factors bind to initiate transcription of specific

genes to mRNA The promoter acts as the regulator for the level of gene expression. Promoters are located near the genes they regulate, on the

same strand and typically upstream (towards the 5' region of

the sense strand).

 Cis-acting elements - DNA sequences in the vicinity of the structural portion of a gene that are required for gene expression Trans-acting factors - factors, usually considered to be proteins, that bind to the cisacting sequences to control gene expression

Eukaryotic Promoters

CAP site

TAC

Core promoters:
Core promoters are Minimal promoters that contain TATA box. The TATA box is also called Goldberg-Hogness box. These DNA sequences are (cis-elements) found in archaea and eukaryotes TATA box is essential for RNA polymerase IIdependent transcription Located at about -20 t0 -35 bp upstream from transcription start site.

Types of Promoters
Constitutive promoters:
Inducible promoters: Tissue-specific promoters :

Synthetic promoters:

Constitutive promoters
They 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 Opine promoters CaMV 35S promoter Monocot promoters Plant ubiquitin promoter (Ubi) Rice actin 1 promoter (Act-1) Maize alcohol dehydrogenase 1 promoter (Adh-1)

Inducible promoters
which are only expressed under the presence of factors /compounds. 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

Tissue Specific 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:
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.

1.Promoters and UAS from opine synthase genes: Patents granted in the U.S. and Australia directed to promoters and Upstream Activating Sequences (UAS) derived from

opine synthase genes, specially from the ocs and mas genes of
A. tumefaciens; 2. 35S-enhanced mas promoter: Patents granted in the U.S. and Canada directed to a mas promoter enhanced by a UAS from CaMV 35S gene Mannopine synthase (mas)

CaMV 35S Promoter

1. Cauliflower mosaic virus (CaMV) replicate through reverse transcription just like retroviruses, but the viral particles contain DNA instead of RNA.

2. The promoter of the 35S RNA is a very strong constitutive

promoter responsible for the transcription of the whole CaMV genome.

3. Most widely used promoter in plant gene transfer.

4. Drives high level of expression in dicots 5. Its ideal for driving expression of selectable marker genes and in some cases of reporter genes.

Inducible 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- 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. Induction of 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
Inducible Inducible

B. Ethanol C. Steroid

D. Copper

Inducible

A. Tetracycline
Tetracycline is used to either De- Repress Gene Expression

Or Inactivate

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 Transactivator (tTA). tTA binds to operators and induces gene expression in absence of tetracycline When Tetracycline is present , tTA is released and expression ceases.

Alcohol inducer:
The ethanol utilization pathway of the filamentous fungus Aspergillus nidulans is a well-characterized positive operon system.

It controls the cellular response to ethanol and other related

chemicals. 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.

Alcohol inducer:
This system uses Chimeric promoters core 35S promoter

region + promoter containing binding sites of AlcR

Transcription factor from Aspergillus nidulans This allows constitutive expression. 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

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 also 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
Numerous patent and patent applications are directed to steroid-responsive promoters for the modulation of gene expression in plant and animal cells McGill University patents on promoters based on the Rat Glucocorticoid Receptor (GR); Rockefeller University patents on promoters based on the Human Estrogen Receptor (ER);

Copper Inducible
This system is based on Metallothionein genes from yeast. Upon binding Copper, the transcription factor binds to Chimeric Promoter and activates the expression. Promoters derived from Metallothionein genes from yeast, mouse and human are the subject matter of several United States patents granted to Genentech and University of California

2. Plant derived systems that respond to environmental signals

Wound Heat

inducible

shock inducible

1. 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,

suggesting that 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 fro expression of pest resistant genes
http://myip.cctec.cornell.edu/techdocs/D1155.D1155_Wound_Inducible_Promoter_ Tech_Brief.pdf

2. Heat shock inducible

HEAT SHOCK ELEMENT(HSE) MEDIATED HEAT SHOCK INDUCIBILITY Soybean heat-shock promoter Gmhsp17.3 Temp. shock of - 400 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.archivesouvertes.fr/docs/00/11/95/62/PDF/BBRC_HAL.pdf)

3. Plant derived systems based on developmental control of gene expression

Senescence

Specific Gene Expression Gene Expression

ABA Inducible 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), an inexpensive, non-toxic,

natural substance can be used as a highly effective chemical inducer ABA is mediates a wide variety of stress responses in plants and induces abscission in leafs during the fall season of the year. Eg: The promoter of the barley ABA-responsive HVA22 gene

Functional dissection of an abscisic acid (ABA)-inducible gene reveals two independent ABA-responsive complexes each containing a G-box and a novel cis-acting element. (http://www.ncbi.nlm.nih.gov/pubmed/7734964)

Auxin Inducible Gene Expression

Auxin plays an essential role in a wide variety of plant growth

and developmental processes, such as shoot and lateral root formation, apical dominance, tropism, and 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

Reference:
1. Plant Biotechnology-The genetic manipulation of plants by Adrian Slater and others 2. Introduction to Plant Biotechnology, by H. S. Chawla 3. Cellular And Biochemical Science edited by G. Tripathi