Download 3-1Basic Bacteriology-Part-III-1

Basic Bacteriology
Part III-1
(UPDATE)
Basic Bacterial Genetics
Dr Alaeddin Abuzant
[email protected]
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Prokaryotic Gene
Structure, Expression and
Protein Secretion
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Bacterial Gene Structure
The genetic information (the codons) of bacterial genes is normally
continuous. That is to say, there are no introns in bacterial genes (in most
cases)
On the other hand, the genetic information of eukaryotic genes (codons that
are found in the exons) are interrupted by non-coding regions/sequences
known as introns.
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For a DNA region to be recognized as a gene it has to have:
1- Coding region/sequence (contains the codons)
2- Regulatory region(s): these are non coding sequence(s) such as the promoter to which RNA
polymerase binds.
Other regulatory DNA regions include enhancer and operator DNA regions, to which regulatory
proteins (activator proteins and repressor proteins) bind to affect the activity of RNA
polymerase positively or negatively, respectively.
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Gene expression: For a gene to be expressed, the gene has to be transcribed by RNA polymerase.
This will generates mRNA which is then get translated by the ribosomes to generate a polypeptide.
The generated polypeptide after that undergoes s folding and may be other post translation
modification to give rise to a functional protein.
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Transcription:
Primary transcripts of eukaryotic gene contains exons and introns
RNA processing: splicing to remove the introns.
mRNA: ligation of exons (in addition 5 caping and poly A tailing)
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Alternative exons ligation: upon removal of introns from the primary transcript, the generated exons
can be ligated in different arrangements so that more that one mRNA may be formed. Each of the
formed mRNAs has a different sequence of the codons so that each of them can give a different protein
upon translation.
Accordingly: an eukaryotic gene may give rise to more than one mRNA and thus more than one protein
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Primary transcript of prokaryotic gene:
Upon transcription of a prokaryotic gene, the generated RNA does not undergo
processing because there are no introns. Accordingly, this trasncript can function as
mRNA.
Each prokaryotic gene give rise to one mRNA, and thus one protein is produced upon
the translation of the prokaryotic mRNA
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In prokaryote, a gene (known also as operon) may have only one coding sequence or more than one
coding sequence under the control of one promoter
On the other hand, upon transcription of a prokaryotic gene, the generated mRNA does not
undergo processing because there are no introns. Accordingly, each gene give rise to one mRNA, and
thus one protein is produced upon the translation of mRNA
Monocistronic operon; has one coding sequence
Upon transcription of monocistronic operon, the generated mRNA is called monocistronic mRNA
because it has one the codons for one protein
Upon translation of the monocistronic mRNA only one protein is generated.
Polycistronic operon: two or more coding sequences are found to be under the control of one
promoter.
Upon transcription of polycistronic operon, the generated mRNA is called polycistronic mRNA
since it contains codons for more than one proteins.
Upon translation of the polycistronic mRNA, (a big) polypeptide is generated that is cleaved to
generate two or more proteins. In many cases, the generated proteins have related functions.
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Note: In genetics, an operon is a functioning unit of genomic DNA containing a cluster
of genes under the control of a single promoter.[1] The genes are transcribed together into
an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to
create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each
encode a single gene product. The result of this is that the genes contained in the operon are
either expressed together or not at all. Several genes must be co-transcribed to define an operon.[2]
Originally, operons were thought to exist solely in prokaryotes, but since the discovery of the first
operons in eukaryotes in the early 1990s,[3][4] more evidence has arisen to suggest they are more
common than previously assumed.[5] In general, expression of prokaryotic operons leads to the
generation of polycistronic mRNAs, while eukaryotic operons lead to monocistronic mRNAs
http://en.wikipedia.org/wiki/Operon
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Polyribosomes
Transcription and Translation Happen
Simultaneously in Bacteria
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Protein Maturation and Secretion
To be functional as a protein, the generated polypeptide:
– requires folding
– association with other proteins and other compounds or metals
– delivered to proper subcellular or extracellular site
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Protein Translocation and
Secretion Systems
• Numerous protein secretion pathways have been
identified
– some reside in all 3 domains
– some unique to Bacteria and Archaea
– some unique to gram-negative cells
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Protein Translocation and Secretion in Bacteria - 2
Translocation: is the movement of proteins from cytoplasm to plasma membrane or
periplasmic space.
Examples: include transport proteins, ETC proteins, proteins involved in chemotaxis and
cell wall synthesis, enzymes.
Two translocation system are known, the Sec translocation system and the Tat translocation
system.
Secretion : is movement of proteins from the cytoplasm to external environment
Examples: hydrolytic enzymes for nutrient break down, toxins……
Six secretion systems have been recognized ( type I to Type VI)
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Translocation Systems
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Secretion Systems
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Regulation of Gene
Expression
Prepared by Dr Alaeddin Abuzant, PhD Microbiology and Immunology
Email: [email protected]
According to their expression: genes are classified into:
1- Constitutive genes ( house keeping genes): these genes are
expressed all the time since their protein products are always
needed
2- Regulated genes: are those genes that their expression is
turned on and off depending whether these genes are needed or
not at a particular situation
To say that a gene is being expressed, this implies that the
following events have to happen:
1- Transcriptions: the gene is transcribed to mRNA by RNA
polymerase
2- Translation: mRNA must be translated into a polypeptide
3- Post-translation events: The produced polypeptide undergoes
folding, could be associated with other proteins, CHO or a metallic
ion could be added or any other modification.
The expression of regulated genes can be controlled at different
levels of gene expression:
A- At the level of transcription
1- Transcription initiation
2- Transcription elongation
B- At the level of translation
1- Translation initiation
2- Translation elongation
C- At the level of post-translational modifications:
Regulated genes can be classified into:
I- Inducible genes: most of the time, the expression of these
genes is tuned of, but some times, their expression in needed to be
turned on (Off→On).
The expression of these genes usually happens upon the
availability of a substrate that the bacterium can utilize, such as
lactose.
In this case, the product of these genes upon their expression is/are
an enzyme or several enzymes that are involved in the degradation
of this substrate.
Accordingly, the gene product(s) of inducible genes are involved
in a catabolic pathway.
II- Repressible genes: most of the time, the expression of these
genes is tuned on, but sometimes, their expression is needed to be
turned off (On→Off).
Since the expression of these genes is ON most of the time, this
implies that product of these genes is/are an enzyme(s) that are
involved the biosynthesis of a compound that is needed most of the
time such as an amino acid. Accordingly, these genes are involved in
an anabolic pathway (biosynthetic pathway)
When the produced compound accumulates and its amount becomes
more than what is needed or when this compound in supplied to the
culture medium in an excess amount, the expression of these genes is
turned off.
The Expression Regulated Genes Is Controlled By:
1- Regulatory DNA regions such as operator site/region and
activator site/region (Enhancer site)
2- Regulatory proteins that bind to these regulatory DNA regions
(operator, activator) to affect the transcription level (increase or
decrease transcription).
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Regulatory Proteins:
Binding of these proteins to regulatory DNA regions either:
1- Activator proteins (activate transcriptions) (have a positive
effect on transcription): these proteins are called activator proteins.
These proteins usually binds to the Enhancer DNA region of a
regulated gene
2- Repressor proteins that repress (stop/inhibit transcription) (
have a negative effect of transcription): these proteins are called
repressors. These proteins usually binds to the operator DNA region
of a regulated gene.
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Regulatory Proteins can be found either in an Active or
Inactive forms:
As mentioned previously, repressor proteins ( repress/inhibit/stop)
transcriptions through bonding to the operator region of a regulated gene.
Active repressor: When ever the repressor protein is able to bind by itself
to the operator region, this repressor is said to be active ( active repressor
protein). To be removed/detach from the operator region, it needs a help.
This can be seen in case of inducible genes (off most of the time) that
is/are regulated an operator region and active repressor protein.
Inactive repressor: In some cases, the repressor protein is NOT able to
bind by itself to the operator region and it needs help to do so.
This repressor protein is said to be inactive ( inactive repressor protein).
This can be seen incase of repressible genes (On most of the time) if these
genes
30 are regulated by an operator region and Inactive repressor protein.
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As mentioned previously, activator proteins (activate transcriptions) bind
to the Enhancer DNA region of a regulated gene to promote or initiate
trasncription.
Active activator: When ever the activator protein is able to bind by itself
to the enhancer region, this activator is said to be active ( active activator
proteins). To be removed/detach from the enhancer, it needs a help. This
can be seen in case of repressible genes (on most of the time) that is/are
regulated an enhancer region (example, tryptophan operon) and an active
activator protein..
Inactive activator: In some cases, the activator protein is NOT able to
bind by itself to the enhancer region. It needs a help to be able to bind.
This activator is said to be inactive ( inactive activator protein). This can
be seen incase of inducible genes (off most of the time) if these genes are
regulated by an enhancer region and inactive activator protein.
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An example of Inducible geneses:
Inducible genes usually encode for enzymes that are involved in catabolic
pathways. In other words, these inducible are repressed most of the time (off
most of the time) and are expressed only when their substrate (to be
degraded or hydrolyzed) of these catabolic enzymes is available.
This substrate (to be degraded or hydrolysed) is known as inducer
Example of inducible genes: The lac operon:
In the absence of lactose, the lac operon is repressed. Induction of lac operon
transcription occurs only when lactose is available.
One of the genes of this operon is β-galactosidase gene that encodes for βgalactosidase. This enzyme catalyzes lactose hydrolysis into galactose and
glucose
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Off → On
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Regulation of the lac Operon by the
lac repressor (Lac I)
Lactose can be called
as an inducer
Inducible Operon
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An example of repressible genes:
Repressible genes usually encode for enzymes that are involved in anabolic
pathways that synthesize a particular important substance for the cell.
In other words, these repressible genes are expressed all the times (On most
of the time) except when the concentration of the substance involved in its
synthesis becomes more that what is needed.
In this case, the high concentration of the produced substance has a negative
impact of the expression of genes involved in its synthesis.
Example of inducible genes: The tryptophan operon
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On → Off
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Regulation of the trp Operon by Tryptophan and
the trp Repressor
Tryptophan can be called as
co-repressor
Repressible Operon
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Genes in bacteria are also called operon, Which could be:
Monocitronic (one coding sequence under the control of
one promoter
Polycictronic: several coding regions that are regulated
by one promoter
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Specialized Nomenclature
Regulon: genes or operons controlled by a common
regulatory protein
Modulon: operons network under control of a common
global regulatory protein but individual operons are
controlled separately by their own regulators
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Regulation of the Lactose
Operon Expression
(Lac Operon)
(In Reality)
The Lactose Operon ( lac operon) :
This operon is involved in lactose utilization. It is an inducible operon that only
expressed when lactose is available to bacterial cells.
This implies that most of the time, this operon is not expressed and it is only
expressed when lactose is available. The Lac operon has three coding (lac z, lac
y, & lac a)
lac z encodes for Beta- Galactosidase that degrades lactose into glucose and
agalactose
lac y encodes for a Permeas or a lactose transporter
lac a encodes for Transacetylase an enzymes involved in galactose utilization
Regulatory DNA regions of the lac operon includes:
1.
A n enhancer region
2.
A promoter
3.
An operator
Regulatory proteins involved in the regulation of the lac operon|:
Lac I: which is a repressor ( an active repressor) that binds to the operator region
spontaneously
CAP: which is an inactive activator protein ( inactive activator) that binds to the
enhancer region only when c-AMP is bound to it. Binding of cAMP to CAP activates it
and makes it able to bind to the enhancer region
Upon the presence of lactose, it binds to Lac I repressor causing lac I to leave the
operator DNA region.
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Note:
In the absence of glucose, cAMP concentration is high so that cAMP
binds to CAP and activates it and makes it able to bind to the
enhancer region.
Upon the presence of glucose, Glucose causes the concentration of
cAMP to decrease , so that there will be no enough cAMP to bind to
CAP. In this case, CAP will detach from the enhancer region.
The effect of Glucose on the expression of the lac operon is called
CATABOLIC REPRESSION
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Expression of Lactose Operon is affected lactose and Glucose
I- Both lactose and Glucose are absent
When lactose is absent, lac I ( active repressor) binds to the operator site
spontaneously
When glucose is absent, the concentration of cAMP is high. So, it will bind to the
CAP (inactive activator) and helps it to become active and thus to become able to bind
to the enhancer region
In this case
CAP-cAMP binds to the Enhancer region
BUT Lac I binds to the operator region
CAP-cAMP activates transcription
BUT
The net effect:
Lac I suppresses transcription
I- Both lactose and Glucose are present
The presence of glucose causes the concentration of c-AMP to decrease.
In this case, the concentration of cAMP is low and thus c-AMP will NOT bind to CAP.
CAP alone without C-AMP will detaches (leaves ) the enhancer region because by its
self, CAP is an inactivate activator
In the presence of Lactose, lactose (allolactose) binds to Lac I causing it to leave the
operator region ( remember that lac-I alone is an active repressor, it binds sponatenously
to the operator site. The binding of lactose to it will makes it inactive and thus it will
detaches from the operator region
The net effect:
No activation of transcription occurs (CAP is not now bound to the enhancer region)
No inhibition of transcription occurs (Lac I is not bound to the operator)
Although these is no inhibition of transcription, there is no activation of transcription as
well. Accordingly, the net effect will be
NO TRANCRIPTION HAPPENS
III- Lactose is present but Glucose is absent
In the absence of glucose , c-AMP concentration becomes high, thus, there will be enough
cAMP to bind to CAP (CAP alone is an inactive activator and binding of cAMP to it will
activates it). In this case, CAP-cAMP will binds to the enhancer region to activates
transcription
Recall that, lac I is an active repressor, so it means it binds to the operator by it self
resulting in the inhibition of transcription. Since lactose is present, lactose will bind to Lac
I, causing Lac I to become inactive and to leave the operator region . This means the
transcription process is free to start
Lactose is present but Glucose is absent means that:
cAMP-CAP is bound to the Enhancer region------this will activate transcription
Lac I is not bound to the operator -------- No inhibition of transcription
Net effect: transcription will start
IV- Lactose is absent but Glucose is present
In the presence of glucose , c-AMP concentration deceases. So, there will be no enough cAMP to bind to CAP. Thus CAP will not be able alone (without cAMP, CAP in not active)
to bind to the enhancer region. Thus no activation of transcription occurs.
Recall that, lac I is an active repressor, so it means it binds to the operator DNA region by
it self resulting in the inhibition of transcription. Since lactose is not present ( absent), Lac I
will continue to bind to the operator region to inhibit the transcription process
Lactose absent but Glucose is present means that:
Lac I is bound to the operator resulting in inhibition of transcription since Lac I is a
repressor
CAP will NOT be bound to the enhancer region, thus, there will be no activation of the
transcription process.
Net effect: transcription will not start