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E.coli promoters
1. E. coli Promoters and its types
SEMINAR ON :
SHWETHA C Y
MSc Biotechnology
2nd YEAR ( 3rd SEMESTER)
BTH- 301 Genetic Engineering
2. Contents discussed
• Introduction to promoters
• Definition of Promoter
• Types of promoter
• E. coli Promoters
• Ribosomal binding sites
• Codon selection
• Conclusion.
3. Introduction :
The flow of genetic information in the cell starts
at DNA, which replicates to form more copies
DNA. Information is then transcribed into RNA,
and then its translated into proteins.
For this complete process of gene regulation to
take place there is a need of regulatory
elements such as ‘ PROMOTERS’ .
4. WHAT ARE PROMOTERS ?
Promoter is a region of DNA that initiates
transcription of a particular gene, Located near
the transcription start sites of genes, on the
upstream region on the DNA(towards the 5').
Promoters can be about 100–1000 base pairs
long.
5. Promoters regulates gene transcription by
allowing RNA polymerase and transcription
factors to bind to it.
In genetic engineering, the promoter is, thus, an
important element in expression vectors driving
the expression of target genes.
6. Promoter is the critical component of
an expression vector
Because the promoter controls the very first
stage of gene expression ( attachment of an RNA
Polymerase enzyme to the DNA ) and
determines the rate at which mRNA is
synthesized.
The amount of recombinant protein obtained
therefore depends to great extent on the nature
of the promoter carried by the expression
vector.
7. Promoter must be chosen with care:
Promoters sequence are consensus sequences
and a small variation may have a major effect on
the efficiency with which the promoter can
direct transcription.
8. Two Types of promoters
1. Strong Promoter
2. Weak Promoter
• Strong Promoter : These are those that can
sustain a high rate of transcription products
are required in large amount by the cell.
• Weak promoter : These are relatively
inefficient , direct transcription of genes
whose products are needed in only small
amounts.
9. E.coli Promoters
E.coli promoters has 3 conserved elements i.e.
-35 sequence : centred 35 base pairs upstream
of the start point of transcription , this sequence
element has both the consensus sequence
‘ TTGACA ’
-10 sequence : Centred 10bps upstream of the
start point of transcription, this sequence
element has the consensus sequence
‘ TATAAT’
10. Spacer : The distance between the above two
conserved elements is also important and is
conserved at 17+/- 1 base pairs.
E coli’s RNA Pol is a multi subunit enzyme.
The core enzyme consist of two identical alpha
subunits and one each beta and beta’ subunits.
11. RNA Pol recognizes different types of promoters
depending on type of sigma factor.
The most common is sigma 70 , which consists
of consensus sequence with -35 and -10 region,
and RNA pol bind to both the sequence to
indicate the transcription.
12. DIFFERENT TYPES OF E coli promoters :
1. Lac Promoter
2. Trp Promoter
3. Tac Promoter
4. Lambda P1 Promoter.
13. Lac Promoter
This is the sequence that controls the
transcription of Lac Z gene coding for
Beta- Galactosidase,
The Lac Promoter is induced by
IPTG(isopropylthiogalactosidase) and addition of
this chemical into growth medium switches on
transcription of gene of the lac promoter.
14. Role of promoter with Lac
The metabolism of lactose in E.
coli & the lactose operon :
To use lactose as an energy
source, cells must contain the
enzyme b-galactosidase.
Utilization of lactose also
requires the enzyme lactose
permease to transport lactose
into the cell.
Expression of these enzymes is
rapidly induced ~1000-fold
when cells are grown in lactose
compared to glucose.
15. LacZ: b- galactosidase;
Y: galactoside permease;
A: transacetylase (function unknown).
P: promoter;
O: operator.
LacI: repressor; PI and LacI are not part of the operon
IPTG: non-metabolizable artificial inducer (can’t be
cleaved)
19. • Negative regulation: The
product of the I gene, the
repressor, blocks the
expression of the Z, Y, and
A genes by interacting
with the operator (O).
• The inducer (lactose or
IPTG) can bind to the
repressor, which induces a
conformational change in
the repressor, thereby
preventing its interaction
with the operator (O).
When this happens, RNA
polymerase is free to bind
to the promoter (P) and
initiates transcription of
the lac genes.
20. Trp Promoter
This promoter is normally upstream of the
cluster of genes coding for several of the
enzymes involved in biosynthesis of the amino
acid tryptophan. The trp promoter is repressed
by tryptophan.
21.
22. The trp operon: two kinds of negative regulation
Tryptophan + trp repressor dimer
Trp-repressor complex
activated for DNA binding
Binds Operator; blocks
RNAP binding &
represses transcription;
Tryptophan a co-
repressor
23. Tac Promoter
The Tac-Promoter (Ptac) or tac vector is a
synthetically produced DNA promoter, produced
from the combination of promoters from the
trp and lac operons(Hybrid).
Commonly used for protein production in
Escherichia coli, Pichia pastoris and in analytical
operation processes like molecular cloning.
24. The tac promoter is used to control and
increase the expression levels of a target gene
and is used in the over-expression of
recombinant proteins.
25. Lambda P1 Promoter
This promoter is one of the promoter
responsible for transcription of the lambda DNA
molecule, which is very strong promoter that is
recognized by the E.coli RNA pol.
Promoter pR and pRM are the bacteriophage
lambda promoters that direct transcription
30. Ribosome Binding region
A ribosome binding site, or ribosomal binding
site (RBS), is a sequence of nucleotides
upstream(5’) of the start codon of an mRNA
transcript that is responsible for the recruitment
of a ribosome during the initiation of protein
translation.
31. The RBS in prokaryotes is a region upstream of the
start codon. This region of the mRNA has the
consensus 5'-AGGAGG-3', also called the
Shine-Dalgarno (SD) sequence.
The complementary sequence (CCUCCU),
called the anti-Shine-Dalgarno (ASD) is contained in
the 3’ end of the 16S region of the smaller (30S)
ribosomal subunit. Upon encountering the
Shine-Dalgarno sequence, the ASD of the ribosome
base pairs with it, after which translation is
initiated.
32. The rate of translation depends on
two factors:
1. The rate at which a ribosome is recruited to
the RBS.
2. The rate at which a recruited ribosome is able
to initiate translation.
33. RBS in eukaryotes
• Ribosome recruitment in eukaryotes happens when
eukaryote initiation factors elF4F and poly(A)-binding
protein (PABP) recognize the 5' capped mRNA and
recruit the 43S ribosome complex at that location.
• Translation initiation happens following recruitment of
the ribosome, at the start codon found within the
Kozak consensus sequence ACCAUGG.
• Since the Kozak sequence itself is not involved in the
recruitment of the ribosome, it is not considered a
ribosome binding site.
35. Codon:
Codon is a series of three nucleotides (triplet)
that codes a specific amino acid residue in a
polypeptide chain, for the initiation as well as
termination of translation.
36. WHY DO CODON MATTER ?
• Because of Redundancy in the genetic code
• Due to mutation effect in protein expression
and rate varying up to 1000 folds
• And also the mutation can alter
Protein conformation, Post Translational
modification, Stability and Function
37. Thus codon selection and usage is an essential
feature of all genomes., which has a main role in
gene expression and impact with the process of
translation.
Optimization of codons has been used to
enhance the protein expression in a genome
38. Conclusion :
Promoters are defined as the DNA sequence
immediately surrounding the transcription start
site, which is bound by the basal transcription
machinery and allows for the initiation of
transcription
In genetic engineering, the promoter is, thus, an
important element in expression vectors: driving
the ‘expression of target genes’.
39. Ribosome binding site is a sequence of
nucleotides upstream of the start codon of an
mRNA transcript that is responsible for the
recruitment of a ribosome during the initiation
of protein translation.
Codon selection is an essential feature of all
genomes and have a main impact on translation
also with its gene expression for a desired
product.
