The document discusses prokaryotic expression systems, which are genetic constructs used to produce proteins or RNA inside or outside cells. It focuses on the prokaryotic system, which is most widely used for recombinant protein production in E. coli bacteria. Key aspects covered include the history of prokaryotic expression research, types of gene regulation like operons and regulatory molecules, examples of operons like the lac and trp operons, and differences between prokaryotic and eukaryotic gene expression.

1. Prokaryotic expression system
DEPARTMENT OF MICROBIOLOGY
THE UNIVERSITY OF HARIPUR

2. Introduction
Expression System
Expression systems are genetic constructs (a gene encoded
by DNA) that are designed to produce a protein, or an RNA
(ribonucleic acid), either inside or outside a cell. Expression
systems are used in research and in the commercial production of
enzymes or therapeutics.
 For overproduction of recombinant proteins both eukaryotic and
prokaryotic expression systems are used.

3. Definition
The most widely used system for protein
overproduction, both on a laboratory and
industrial scale, is the prokaryotic system.
This system is based primarily on the bacteria
E. coli, although increasingly often Bacillus
species are used.

4. History
 1962 the concept of bacterium (Roger Stanier and C.B van Niel).
 1977 Carl Woese proposed divide prokaryotes in archaea and
bacteria.
 Lac operon model was discover by François Jacob and Jacques
Monod in 1961.
 Won Nobel Prize in 1965.
 In 1977 Herbert boyer and Arthur riggs described the first vector
pBR322-plasmid widely used E. coli cloning vectors.

5. Types and components of prokaryotic gene regulation
Operon
Trp Operon: A repressor operon
Catabolite Activator Protein(CAP):An
activator regulator

6. Prokaryotic Gene Regulation
 The DNA of prokaryotes is organized into a circular
chromosome supercoiled in the nucleoid region of the
cell cytoplasm. Proteins that are needed for a specific
function are encoded together in blocks
called operons.
 For example, all of the genes needed to use lactose as
an energy source are coded next to each other in the
lactose (or lac) operon.

7.  In prokaryotes, structural genes of related function are often organized together on the
genome and transcribed together under the control of a single promoter. The operon’s
regulatory region includes both the promoter and the operator. If a repressor binds to the
operator, then the structural genes will not be transcribed. Alternatively, activators may bind
to the regulatory region, enhancing transcription.

8. Types of gene regulation
 There are two different types of gene regulation: positive and
negative. Activators (and sometimes inducers) instigate
positive regulation, and repressors instigate negative
regulation. When an activator or inducer binds to an operon,
the transcription process either increases in rate or is allowed
to continue. When a repressor binds to an operon, the
transcription process is slowed or halted.

9. Types of regulatory molecules
 In prokaryotic cells, there are three types of regulatory molecules that can affect
the expression of operons: repressors, activators, and inducers.
 Repressors are proteins that suppress transcription of a gene in response to an
external stimulus. In other words, a repressor keeps a gene “off.”
 Activators are proteins that increase the transcription of a gene in response to
an external stimulus. In other words, an activator turns a gene “on.”
 Inducers are small molecules that either activate or repress transcription
depending on the needs of the cell and the availability of substrate. Inducers
basically help speed up or slow down “on” or “off” by binding to a repressor or
activator. In other words: they don’t work alone.

10. Cistron
 The cistron is the genetic unit coding for the structure of the subunit of a
protein molecule.
 A single mRNA carries information for multiple proteins.
 This type of mRNA is called a polycistronic mRNA and is totally unique to
prokaryotes.
 The polycistronic Lac Operon mRNA is translated into three separate
proteins.
 In Eukaryotes the m-RNA is monocistronic.

11. The lac Operon: An Inducer Operon
 The lac operon (lactose operon) is an operon required for the
transport and metabolism of lactose in Escherichia coli and
many other enteric bacteria. Although glucose is the preferred
carbon source for most bacteria, the lac operon allows for the
effective digestion of lactose when glucose is not available
through the activity of beta-galactosidase
 The type of gene regulation in prokaryotic cells occurs
through inducible operons.
 The lac operon is a typical inducible operon.
 The lac operon encodes the genes necessary to acquire and
process the lactose from the local environment.

12. Components of Lac Operon

13. In Absence of lactose
 The lac repressor bind to DNA sequence called the operator
region( found btw lac z and the promotor)
 In this way the lac repressor block the path of RNA polymerase to
reach the lac z y and a gene and the lac promotor)
 mRNA and proteins cant be formed in the absence of lactose
 It cants even work in the presence of glucose even at low amount

14. In the presence of lac
 Lactose molecules are metabolized by the lac enzyme, an
intermediates are formed called allolectose (isomers of
lactose)
 Allolactose act as inducers by binding to the lactose
repressor by changing its confirmation therefore it can no
longer bind to the operator
 Operon sense the glucose presence and by the mechanism
called catabolite repression it is switched off

15. Condition required for lac operon activation
For the lac operon to be activated, two conditions required.
1. First, the level of glucose must be very low or non-existent.
2. Second, lactose must be present.
 Only when glucose is absent and lactose is present will the lac operon be
transcribed. This makes sense for the cell, because it would be
energetically wasteful to create the proteins to process lactose if glucose
was plentiful or lactose was not available.

16. Transcription of the lac operon is
carefully regulated so that its
expression only occurs when
glucose is limited and lactose is
present to serve as an alternative
fuel source.

17. Trp operon ( negative)
 Tryptophan is one such amino acid that E.coli can ingest from
environment.
 E.coli can also synthesized tryptophan using enzymes that are
encoded by five genes
 Theses five genes are next to each other in what is called tryp
operon

18. How tryptophan works
 If trp is present in environment then E.coli doesn’t need to
synthesized
 The switch controlling the operon will switched off
 If the trp is not present or its availability is low in
environment or in E.coli the switch controlling the operon will
be turned on
 Transcription initiated genes are expressed and the
tryptophan synthesized.

19. Figure. The five genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon.
When tryptophan is plentiful, two tryptophan molecules bind the repressor protein at the operator sequence. This physically
blocks the RNA polymerase from transcribing the tryptophan genes. When tryptophan is absent, the repressor protein does
not bind to the operator and the genes are transcribed.

20. CAP( AN ACTIVATOR REGULATOR)
 There are also binding protein that stimulate the transcription
 One particular example is catabolite activator protein (cap) which is also known
as cAMP response protein(CRP)
 It is positive regulator.
 When glucose level drop cAMP begin to accumulate in the cell
 cAMP molecule is signaling molecule that is involved in glucose and energy
metabolism in E.coli
 When glucose level drop accumulating camp bind to pos regulator CAP, a
protein that bind to the promoter of operons that control the processing of
alternative sugars

21. Figure. When glucose levels fall, E. coli may use other sugars for fuel but must transcribe new genes to do so. As glucose
supplies become limited, cAMP levels increase. This cAMP binds to the CAP protein, a positive regulator that binds to an
operator region upstream of the genes required to use other sugar sources.

22. Differences
Prokaryotes
 Prokaryotic genes are grouped in operons
 Prokaryotes have one type of RNA polymerase
for all types of RNA.
 mRNA is not modified(polycistronic)
 The presence of introns in prokaryotes is rare.
 In prokaryotes, the newly synthesized mRNA is
polycistronic.
 In prokaryotes, transcription of gene and
translation of resulting mRNA occur
simultaneously.
Eukaryotes
 Eukaryotes genes are not grouped in operon
 Eukaryotes have three different RNA
polymerases responsible for different classes
of RNA molecules.
 mRNA are monocistronic.
 Both intron and exons are present in
eukaryotes.
 mRNA is processed before transport to the
cytoplasm where it is translated.

23. “
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