Class presentation on Isolation of promoters and other regularly elements

1. Isolation of Promoters and other regulatory
elements
MBB: 601 Advances in Plant Molecular Biology
Presented By
Mr. Ekatpure Sachin
PhD Research Scholar
Department of Plant Biotechnology

2. 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.

3. Typical Gene Structure

4. 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

5. 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

6. Simplified model of transcriptional regulation of protein-encoding genes

7. 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

8. 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

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

10. 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

11. Conserved eukaryotic promoter elements
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
• It seems to function in either orientation.
• This box is replaced in plants by a consensus sequence called the AGGA box
TATA 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 factors
GC 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
box
CAP site
• A transcription initiation sequence or start point defined as +1, at which the transcription
process actually starts.

12. Consensus Sequences
Conserved eukaryotic promoter elements Consensus sequence
CAAT box GGCCAATCT
TATA box TATAAAA
GC box GGGCGG
CAP site TAC

13. TYPES OF PROMOTERS USED TO REGULATE
GENE EXPRESSION

14. Types of promoters
Promoters used in
biotechnology
Constitutive
promoters
Tissue specific
promoters
Inducible
promoters
Synthetic
promoters

15. 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

16. 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)

17. 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

18. 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

19. Tissue specific
Promoters
Root
Promoters
Fruit
Promoters
Seed
Promoters

20. 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

21. Inducible Promoters
Chemically regulated
Alcohol
regulated Pathogen related
SA, Ethylene, Thiamine,
Benzol
Steroid regulated
Glucocorticio
d 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

22. 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

23. Methods of Promoter Isolation

24. 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 plant
Plasmid rescue
Inverse PCR (IPCR)
Genome Walking
The thermal asymmetric interlaced PCR (TAIL-PCR)

25. 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

26. 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

27. 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

29. 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

31. 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.

33. 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

34. 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

35. 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)

36. 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)

37. 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

38. Cycling Orders
Liu & Whittier, 1995

39. 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.

40. Thank You