Isolation of Promoters and other regulatory elements

MBB: 601 Advances in Plant Molecular Biology

Presented ByMr. Ekatpure Sachin

PhD Research ScholarDepartment of Plant Biotechnology

Central Dogma of life

The flow of information in the cell starts at DNA, which replicates to form more DNA. Information is then ‘transcribed” into RNA, and then it is “translated”

into protein.

Typical Gene Structure

Definition

Gene promoters are DNA sequences located upstream of gene coding regions and contain multiple cis-acting elements, which are specific binding sites for proteins involved in the initiation and regulation of transcription

In most transcription units, the promoters is located next to transcription start site but is not itself transcribed

Introduction• Promoters often contain a “core promoter”, which is a region located 40 bp ∼

upstream of the transcriptional initiation site which contains the TATA box

• The TATA box is the binding site for the transcription initiation factor TFIID TBP (TATA-box-Binding Protein) subunit

• • The core promoter also contains cis-elements that are binding sites for the basic

transcriptional machinery, including RNA polymerase II and its corresponding subunits

• A protein complex, including general transcription factors such as TFIID and TFIIB is formed with RNA polymerase II prior to initiation of transcription

Simplified model of transcriptional regulation of protein-encoding genes

Cont..

• Upstream of the core promoter region are the proximal and distal regions of promoters

• Proximal and distal regions of the promoter contain different regulatory sequences such as enhancers, silencers, insulators, and Cis-elements that contribute to the fine regulation of gene expression at the transcriptional level

Cont..• During transcription, co-activators and transcription factors bind to specific DNA motifs

and simultaneously interact with the transcriptional machinery attached to the core promoter

• This complex DNA/protein interaction leads to the activation, enhancement, or suppression of transcription

• Thus, regulation of transcription depends on the:

– Availability and activity of transcription factors

– The type, number, position, and combination of regulatory elements present in and around the promoter

cont…

• Regulation of gene expression at the promoter level is mainly controlled by the cis-acting elements localized upstream of the transcriptional start site

Cis- and trans-acting factors regulate gene expression

• The intricate pattern of gene regulation involves molecular signals that act on DNA sequences encoding protein products

• Cis-acting molecules act upon and modulate the expression of physically adjacent, operably linked polypeptide-encoding sequences

• Trans-acting factors affect the expression of genes that may be physically located very far away, even on different chromosomes

• The expression of a particular gene may be regulated by the concerted action of both cis and trans-acting elements

Conserved eukaryotic promoter elementsCAAT 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• It seems to function in either orientation. • This box is replaced in plants by a consensus sequence called the AGGA boxTATA box• A sequence usually 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 factorsGC box• A sequence rich in guanidine (G) and cytosine (C) nucleotides• is usually found in multiple copies in the promoter region, normally surrounding the TATA boxCAP site• A transcription initiation sequence or start point defined as +1, at which the transcription process

actually starts.

Consensus Sequences

Conserved eukaryotic promoter elements Consensus sequence

CAAT box GGCCAATCT

TATA box TATAAAA

GC box GGGCGG

CAP site TAC

TYPES OF PROMOTERS USED TO REGULATE GENE EXPRESSION

Types of promoters

Promoters used in biotechnology

Constitutive promoters

Tissue specific promoters

Inducible promoters

Synthetic promoters

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

Constitutive Promoters

Plant Pathogen Promoters

CAMV 35S Promotes

Opine Promoters

Monocot Promoters

Plant Ubiquitin Promoter (Ubi)

Rice Actin 1 (Act-1)

Maize alcohol dehydrogenase

1 (Adh1)

Advantages• High level of production of proteins used to select transgenic cells or plants;

• 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

Tissue-specific or development-stage-specific 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, so they may be also classified as inducible.

• For plants, promoter elements that are expressed or affect the expression of genes in the vascular system

• Photosynthetic tissues• Tubers• Roots• Other vegetative organs• Seeds• Reproductive organs can be found in heterologous systems

Tissue specific Promoters

Root Promoters

Fruit Promoters

Seed Promoters

Inducible promoters• These are only expressed under the presence of factors/compounds • Because their expression is normally restricted to certain plant tissues

• They can also be considered as tissue-specific

• Based on the nature of the factors that trigger their expression, they are divided into two groups:

– Chemically-regulated: where chemical compounds, usually not naturally found within plants, switch on promoter activity. Several of the types of promoters involves chimeric components gathered from human, animal, fungal and bacterial sources

– Physically-regulated: where abiotic and external factors such as light, heat, mechanical injury induce promoter activity

Inducible Promoters

Chemically regulated

Alcohol regulated Pathogen related

SA, Ethylene, Thiamine, Benzol

Steroid regulated

Glucocorticiod receptors

(GR)

Glucocorticoid response

element (GRE)

Metal regulated

Copper, Zinc, Gold, Mercury,

Cobalt

Tetracycline regulated

Antibiotic resistance

Physically regulated

Temperature regulated

Heat inducible Cold inducible

Light regulated

Light inducible Light repressible

Synthetic promoters

• Synthetic promoters are DNA sequences that do not exist in nature and which are designed to regulate the activity of genes, controlling a gene’s ability to produce its own uniquely encoded protein

Methods of Promoter Isolation

Methods of Promoter Isolation

• There are number of methods are available to isolate the promoters some of them are listed below

Screening of the genomic DNA library constructed from the mutant plantPlasmid rescueInverse PCR (IPCR)Genome WalkingThe thermal asymmetric interlaced PCR (TAIL-PCR)

1. Screening of the genomic DNA library constructed from the mutant plant

• T- DNA and transposable elements were used in the creation of mutant plants

• DNA fragments flanking the T-DNA are identified from the library and used as a probe to isolate the wild type genomic sequence

• T-DNA disrupts the expression of the gene also it acts as a marker for subsequent identification of the mutation

Conti…

• In these methods, large populations of tagged mutants are generated, which can then be screened for insertions in specific genes

• Alternatively, the insertion tags can be individually sequenced and compiled in databases that can be searched for a gene disruption event of interest

• This is becoming easier now as genomic sequences of many insertion sites are becoming available

2. Plasmid rescue• Widely used method for obtaining sequence information about the T-DNA–gDNA

junctions in the recipient genome

• It is an approach which is employed in case the T-DNA construct consists of an antibiotic resistance gene and a bacterial origin of replication (ori)

• The genomic DNA of mutant plant is subjected to complete digestion followed by ligation to circularize all the fragments and transform them into E. coli host

• The plasmids isolated from the E. coli would be analyzed for the presence of T-DNA and the flanking plant DNA sequences

3. Inverse PCR (IPCR)• It amplify the unknown sequence from the known sequence

• Cleavage of genomic DNA by a suitable enzyme followed by ligation of the fragments to facilitate self-circularization

• A set of nested primers derived from the T-DNA border regions are used to amplify the flanking DNA, cloned and sequenced

• Specially useful in the amplifying and identifying flanking sequence of various genomic inserts

4. Genome Walking• Genome walking is a method to isolate flanking genomic segments (e.g. promoter regions) adjacent to a known sequence

• Uncloned genomic DNA is digested with various restriction endonucleases

• Ligated to long suppression adapters

• The desired genomic region is amplified with a primer specific to the outer part of the suppression adapter and a gene-specific primer

• Since the adapters are long and the adapter-specific primer is short (the sufficient ratio is 40 to 20 base pairs)

• the amplification of the whole pool from that single adapter-specific primer is effectively suppressed, and only the fragments of interest are generated during PCR

• Both flanks of the known sequence – downstream as well as upstream – can be amplified: the direction depends exclusively upon the strand specificity of the gene-specific primer.

5. The Thermal Asymmetric Interlaced PCR (TAIL-PCR)

• Makes use of three nested T-DNA-specific primers in one end and a short arbitrary degenerate (AD) primer in the other end

• Three different PCR reactions are performed with these primer sets

• The primary PCR reaction involves different primer annealing temperatures and low and high stringent cycles to facilitate annealing of arbitrary and specific primers, respectively

• This step results into both specific as well as nonspecific amplification of products

• In the next two steps of PCR reactions the non-specific products are eliminated amplifying predominantly the T-DNA flanking genomic DNA

Protocol of TAIL-PCR

SP1 SP2 SP3

AD primer

vector insert nontarget sequence

(A) Primary PCR with SP1 and AD

5 high stringency cycles

1 low stringency cycle

Protocol of TAIL-PCR

10 reduced stringency cycles

2 high stringency cycles(thermal asymmetric)

1 reduced stringency cycle(thermal symmetric)

Nonspecific product(type II)

Specific product(type I)

Nonspecific product(type III)

Product yield:

High or middle(detectable or undetectable)

High(detectable)

Low(undetectable)

TAIL-cycling(12 super cycles)

Protocol of TAIL-PCR

(B) Secondary PCR with SP2 and AD (10 super cycles)• 1000-fold dilution of primary PCR product

Specific product Nonspecific product (type III)

Product yield:

High (detectable) Very low (undetectable)

Protocol of TAIL-PCR

(C) Tertiary PCR with SP3 and AD (20 normal cycles)

• 1000-fold dilution of secondary PCR product

Specific product

Agarose gel analysis

Direct sequencing

Cycling Orders

Liu & Whittier, 1995

Applications of TAIL-PCR

• Isolation of 5’ flanking region of genes

• Isolation of promoter sequences

• Isolation of T-DNA insert junctions

• For genome physical mapping, development of sequence-tagged sites (STS), and analysis of genomic sequences flanking T-DNA, transposon or retrovirus insertions.

Thank You