Transcript Transcription

Fundamentals of Gene
Regulation
The DNA of different tissues and cell types is the
same in a specific organism unlike the RNA and
protein content. Gene regulation must therefore
operate to produce different amounts of different RNA
in different cell types, from the same DNA.
Rekha Jain
Harini Chandra
Master Layout (Part 1)
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This animation consists of 3 parts:
Part 1 – Basics of gene regulation
Part 2 – Gene regulation in bacteria
Part 3 – Gene regulation in eukaryotes
PROKARYOTIC
EUKARYOTIC
DNA
RNA
Ground state: on
Ground state: off
Repressed state: off
Active state: on
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PROTEIN
CENTRAL DOGMA
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Please re-draw figures. Source: Biochemistry by Lehninger, 4th edition (ebook), Genetics by Griffiths,
8th edition (ebook)
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Definitions of the components:
Part 1 – Basics of Gene regulation
1. Gene regulation: The process by which the synthesis of a gene’s mRNA transcript and its
corresponding protein product is controlled or regulated by various signal molecules is
termed as gene regulation. In this process, a cell determines which genes to express
and
when to express them. Regulation of processes is essential to ensure that no wastage of
energy or cellular materials takes place.
2. Single celled organisms: Organisms that have only one cell containing all the organelles
and genetic material within one common compartment are known as single celled
organisms. The bacterial genome has 4000 genes of which only a fraction of them are
expressed at any given time. Moreover, requirements for gene products vary with time such
that some products are required in large amounts while others in smaller quantities.
3. Multi-cellular organisms: Gene regulation is a more complex process in eukaryotic and
multi-cellular organisms that contain more number of cell organelles, each having complex
processes taking place in them. The human genome contains around 35,000 genes, out of
which only a fraction of them are expressed in a cell at any given time. Gene expression
varies in different cell types even though their copy of the genome is identical. Certain
genes, known as housekeeping genes, are expressed in all cells while others are specific
only to certain cell types. For example, the gene for glucagon hormone is expressed only in
pancreatic cells while antibody synthesis genes are continuously expressed in plasma cells.
4. Transcription: Transcription is the process by which information from a double stranded
DNA molecule is converted into a chemically related single stranded RNA molecule by
making use of one strand as the template. Transcription which takes place in the eukaryotic
nucleus is separated in space and time from translation taking place in the cytoplasm.
5. Translation: A process by which the mRNA sequence is read in the form of three letter
codes known as codons to incorporate the corresponding amino acids in the growing
polypeptide chain with the active involvement of rRNA, tRNA and several other enzymes.
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Definitions of the components:
Part 1 – Basics of Gene regulation
6. Different levels of gene regulation: Gene regulation can be carried out at several
levels starting from the synthesis of the mRNA transcript till the degradation of its
corresponding protein product. The various stages include:
a) Synthesis of the primary RNA transcript (transcription)
b) Post-transcriptional modification of mRNA
c) Messenger RNA (mRNA) degradation
d) Protein synthesis (translation)
e) Posttranslational modification of proteins
f) Protein targeting and transport
g) Protein degradation
7. Active state: The state in which the gene is turned “on” and synthesizes its
corresponding mRNA and protein.
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8. Repressed state: The state in which the gene is turned “off” and no transcription
occurs. This could be due to binding of a repressor molecule to the gene.
Part
1,
Step
1:
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IN PROKARYOTES
2
Activator protein
RNA pol
No transcription
Transcription
Repressor
3
RNA pol
Coding region of gene
Ground state: on
Repressed state: off
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Action
5
As
shown
in
animati
on.
Description of the action
First show the figure on the left with the coloured line & the red
oval bound to it. The orange oval must then appear and bind to
the red oval & coloured line as depicted. Once this happens, the
green arrow & label must appear. Next show the figure on right
with coloured region & the yellow shape bound to it. The red
oval must now approach the yellow shape and must move away
as soon as it reaches the shape followed by appearance of the
green arrow with red cross over it.
Audio Narration
In prokaryotes, transcription by RNA
polymerase can take place with the help of an
activator protein. However, in the presence of a
repressor molecule, the binding site for RNA
polymerase is inaccessible due to which
transcription does not occur. In the ground
state, the repressor does not remain bound
because of which the gene is turned “on”.
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Part 1, Step 2:
IN EUKARYOTES
Nucleosome –
chromatin &
histone
2
No transcription
Enhancer
Transcription
factors
Promoter
proximal
element
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Transcription
Ground state: off
Active state: on
Action Description of the action
Audio Narration
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As
shown
in
animati
on.
PLEASE RE-DRAW ALL FIGURES. First show
the two oval structures on top which must then
be zoomed into to show the coloured regions
below. Once this happens the green arrow with
red cross over it must appear. Next show the
figure on the right with its labels. This is followed
by appearance of the arrow & the label
‘transcription’.
Source: Genetics by Griffiths, 8th edition (ebook)
The regulatory site of DNA in eukaryotes remains
inaccessible for binding by transcription machinery due to
which gene expression remains turned off in the ground
state. In the active state, however, the DNA forms a loop
thereby bringing together the promoter proximal element
and enhancer . Interactions of these two elements with
RNA polymerase successfully activates expression of the
gene.
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Master Layout (Part 2)
This animation consists of 3 parts:
Part 1 – Basics of Gene regulation
Part 2 – Gene regulation in prokaryotes
Part 3 – Gene regulation in eukaryotes
promoter
Gene A
Transcription
Translation
Promoter
Operator
Gene
Protein A
Negative regulation
Positive regulation
Activator
Transcription
No transcription
4
Repressor
No transcription
Transcription
5
Gene C
mRNA
2
3
Gene B
Protein B
Protein C
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Definitions of the components:
Part 2 – Gene regulation in prokaryotes
1. Operon: A functioning unit of genomic material that is made up of a cluster of functionally
related genes that are under the control of a single regulatory element. Operon arrangements
are a commonly observed mechanism of gene regulation in prokaryotes and can be either
inducible or repressible.
2. Lac operon: The first system of gene regulation that was understood in E. coli, worked out
by Francois Jacob and Jacques Monod in 1962. The lac operon is negatively controlled by the
lacI repressor and positively regulated by catabolic activator protein (CAP).
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3. Operator: Operators are regions of DNA that are around15 nucleotides long and are
generally located near a promoter element such that they control the access of RNA
polymerase to this region.
4. Promoter: The region of DNA to which RNA Polymerase binds and starts the process of
transcription.
5. Activator: A molecule that enhances the interaction between RNA polymerase and a
particular promoter region, thereby facilitating expression of the gene.
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6. Repressor: A protein that binds to a regulatory region such as an operator, adjacent to a
gene and thereby prevents its transcription by impeding the binding of RNA polymerase.
7. Effectors: Effectors are molecules that affect the binding of activators and repressors to the
operator region of DNA. These can either be inducers, in case of inducible systems, or a corepressors if it is a repressible system.
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Definitions of the components:
Part 2 – Gene regulation in prokaryotes
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8. Inducible system: An inducible system is originally ‘off’ in its ground state and must be
turned on by an effector molecule, which is known as the inducer. In the negative regulation
mechanism, the inducer binds to repressor and prevents it from binding to the operator
region. This allows RNA polymerase to proceed with transcription by binding to the promoter.
In positive regulation mechanism however, the inducer binds to the inactive activator to
produce the active activator molecule which in turn facilitates binding of RNA polymerase to
the promoter to turn on expression.
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9. Repressible system: The ground state in case of a repressible system is ‘on’ and it has
to be turned off by an effector molecule, which is known as the co-repressor. In case of
negative regulation mechanism, the co-repressor binds to the inactive repressor molecule
and activates it, thereby preventing gene expression. In positive regulation, on the other
hand, the co-repressor binds to the activator molecule and prevents its binding to the
promoter region, thereby turning off gene expression.
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Part 2, Step 1:
Inducible system
Positive regulation
2
Inducer
Transcription
No transcription
Active
Inactive
activator
Promoter
Operator
Negative regulation
Gene
Inducer
Transcription
No transcription
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Repressor
Promoter
Operator
Gene
4
Action
Description of the action
An inducible system is ‘off’ in its ground state and must be
turned on by an effector molecule, which is known as the
inducer. In the negative regulation mechanism, the inducer
binds to repressor and prevents it from binding to the
which binds to blue cloud. The cloud must change colour & the text box
operator region. This allows RNA polymerase to proceed
must show ‘active activator’. The red cross must disappear over the
with transcription by binding to the promoter. In positive
green arrow & must say ‘transcription’. Next, the coloured chain on the
regulation mechanism however, the inducer binds to the
right is shown with the yellow hexagon bound to the red region & the
inactive activator to produce the active activator molecule
arrow with red cross over it. The grey box must bind to this yellow figure
which in turn facilitates binding of RNA polymerase to the
& both must be removed along with the red cross.
promoter to turn on expression.
As shown First show the coloured chain with labels on the left followed by binding
of ‘inactive activator’ & appearance of the green arrow with red cross
in
animation. saying ‘no transcription’. Next show appearance of the grey square
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Audio Narration
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Part 2, Step 2:
Repressible system
Positive regulation
2
Co-repressor
No transcription
Transcription
Activator
Negative regulation
Promoter
Operator
Gene
Co-repressor
No transcription
3
Active
Inactive
repressor
Promoter
Operator
Transcription
Gene
4
Action
As shown
in
animation.
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Description of the action
Audio Narration
First show the coloured chain with labels on the left followed by binding The ground state in case of a repressible system is
of ‘activator’ & appearance of the green arrow saying ‘transcription’. Next ‘on’. It has to be turned off by an effector molecule,
show appearance of the green star which binds to ‘activator’ and both
which is known as the co-repressor. In case of
these shapes must together get dissociated & the red cross must appear negative regulation mechanism, the co-repressor binds
over the green arrow saying ‘no transcription’. Next, the coloured chain to the inactive repressor molecule and activates it,
on the right is shown with the violet star bound to the red region along
thereby preventing gene expression. In positive
with the green arrow and ‘transcription’ label. Next the light green star
regulation, the co-repressor binds to the activator
must bind to the violet shape, which must change colour & the text must
molecule and prevents its binding to the promoter
change to ‘active repressor’. A cross must appear over the green arrow
region, thereby turning off gene expression.
with the label ‘no transcription’.
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Part 2, Step 3:
The lac operon: negative control by lacI repressor
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Presence of lactose: de-repression
Absence of lactose: repression
No transcription
Transcription
RNA Polymerase
lacI
Operator
lac Z
lacY
lacA
p
Promoter
No binding
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mRNA
b-galactosidase
Repressor
protein
(monomer)
Transacetylase
Permease
Repressor
Inducer binds
tetramer
repressor protein
Inducer
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Action
Description of the action
First show the blue region giving the strand & then the yellow
As shown circle. 4 yellow circles must come together & then bind to the
in
green region. The violet oval must attempt to move forward but
animation. must be blocked by the yellow circles. The green arrow with red
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cross must appear. Next the heading must change & the blue
square below must bind to the yellow circles which should then
attempt to bind to green region but they should not be able to.
The violet oval then moves across the entire coloured region till
the end & ‘transcription’ heading must appear.
Audio Narration
The lac operon consists of a group of genes that are
responsible for transport and metabolism of lactose sugar in
certain bacteria like E. coli. This operon is under negative
regulation by the LacI repressor protein. In absence of the
inducer, the tetrameric repressor binds to the operator region,
thereby preventing transcription by RNA Polymerase. In
presence of the inducer, the inducer binds to the repressor
protein which then prevents it from binding to the operator and
therefore allows gene expression.
Part 2, Step 4:
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The lac operon: Positive Control by CAP and catabolite repression
Low glucose
cAMP
ATP
2
Glucose
Lactose
Bacterial cells
CAP
cAMP
Cell growth &
division
3
High glucose
ATP
4
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Action
Transcription
lacI
cAMP
Operator
lac Z
lacY
lacA
Promoter
Description of the action
First show the blue region giving the strand & then the yellow
As shown circle. 4 yellow circles must come together & then bind to the
in
green region. The violet oval must attempt to move forward but
animation. must be blocked by the yellow circles. The green arrow with red
cross must appear. Next the heading must change & the blue
square below must bind to the yellow circles which should then
attempt to bind to green region but they should not be able to.
The violet oval then moves across the entire coloured region till
the end & ‘transcription’ heading must appear.
Source: Introduction to Genetic Analysis, eight edition (ebook)
Audio Narration
Lac operon also undergoes positive regulation by means of
the cAMP-CAP system. Glucose is the preferred energy
source for bacteria and if both glucose and lactose are
present, b-galactosidase enzyme which metabolizes lactose
is not synthesized. High glucose levels prevent synthesis of
cAMP which is essential for binding to the catabolite activator
protein. This protein facilitates transcription of the lac operon.
When glucose levels are low, cAMP is produced which binds
to the CAP, which in turn binds to a distal part of the
promoter region and facilitates transcription.
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Master Layout (Part 3)
This animation consists of 3 parts:
Part 1 – Basics of Gene regulation
Part 2 – Gene regulation in bacteria
Part 3 – Gene regulation in eukaryotes
2
Inaccessible state
Gene
DNA
(1) Transcription
Primary transcript
(2) Post-transcriptional
processing
Mature mRNA
3
Chromatin remodeling
4
(3) mRNA
degradation
(4) Translation
Protein
(inactive)
Accessible state
Nucleotides
(5) Post-translational
processing
Amino acids
(7) Protein
degradation
Modified
protein
(active)
(6) Protein targeting
and transport
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Please re-draw all figures. Source: An introduction to Genetic analysis by Griffiths, 8th edition (ebook)
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Definitions of the components:
Part 3 – Gene regulation in eukaryotes
1. Gene expression: The process of transfer of genetic information from the nucleotide
sequence level in a gene to the level of amino acid sequence in a protein or the nucleotide
sequence of mRNA is known as gene expression.
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2. Eukaryotic regulation: Eukaryotic cells have larger and more complex multimeric
regulatory proteins when compared to bacterial cells. The process of regulation is therefore,
also more complex and can be achieved either by altering the rate of transcription, the
stability of mRNA molecule or through regulation at a translational level. Regulatory elements
that control these processes may be tissue specific, thereby activating or deactivating genes
only in one kind of tissue.
3. Chromatin: DNA that is packaged with basic proteins known as histones form a structure
known as chromatin in eukaryotes. This chromatin structure helps in restricting access to
eukaryotic promoter sites. For gene expression to take place, remodelling of the chromatin
must occur wherein, acetylation of histone proteins and demethylation of DNA occur, which
then favours transcription.
4. Promoter: The region of DNA to which RNA Polymerase binds and starts the process of
transcription. The promoter in eukaryotes contains a sequence of 7 bases known as the
TATA box, which is bound by a large number of proteins including the TATA-binding protein
(TBP), and various transcription factors.
5. Exons: The regions of mRNA that code for specific proteins or entire protein products upon
translation are known as exons. They are often discontinuous with intervening nucleic acid
sequences being present between them.
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6. Introns: The intragenic sequences, sometimes considered as “junk”, that are present in
the pre-mRNA but do not get translated into proteins are known as introns. These are
removed during the process of RNA splicing.
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Definitions of the components:
Part 3 – Gene regulation in eukaryotes
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7. Enhancers: Enhancers are sequences of DNA to which regulatory proteins can bind.
Most of these are located outside of the promoter region. Binding of transcription factors to
enhancers is associated with an increase in the rate of transcription.
8. Silencers: Silencers are control regions of DNA, onto which transcription factors bind in
order to decrease the rate of transcription.
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9. RNA splicing/post-transcriptional modification: It is the process by which exons are
spliced or cut out from the pre-mRNA molecule to give a coding, mature mRNA sequence,
which is then translated into protein. This is another point at which gene regulation
commonly occurs. Splicing can take place such that the exons are re-joined in different
combinations, in a process known as alternative splicing. This allows a variety of different
polypeptides to be translated from a single gene.
10. RNA Transport: The process by which only fully processed and mature mRNA is
allowed leave the nucleus in order to be translated into protein. Any defective mRNA will be
degraded within the nucleus itself.
11. Positive regulation: Although eukaryotic cells exhibit both positive and negative
regulatory mechanisms, the positive mechanisms have been found to predominate in all
systems characterized so far. The transcriptional ground state is therefore restrictive or
silenced, and virtually every eukaryotic gene requires activation before it can be transcribed.
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Part 3, Step 1:
Gene
DNA
(1) Transcription
2
Primary transcript
(2) Post-transcriptional
processing
Nucleotides
(3) mRNA
degradation
Mature mRNA
3
(4) Translation
(7) Protein
degradation
Amino acids
(6) Protein targeting
and transport
(5) Post-translational
processing
4
Action
As shown
in
animation.
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LEVELS OF REGULATION OF
EUKARYOTIC GENE
EXPRESSION
Description of the action
PLEASE RE_DRAW ALL FIGURES. First
show the figure on top appearing followed
by the various down arrows and the figures
below each arrow. Then show all the
backward arrows as depicted in animation
above.
Source: Biochemistry by Lehninger, 4th edition (ebook)
Audio Narration
Eukaryotic gene regulation is a complex process that
can be regulated at various levels starting from the
gene transcription to the post-translational
modification of the protein to form the active,
functional molecule. These levels include transcription,
post-transcriptional modification, translation, posttranslational modification and protein transport, the
most common of which is the transcriptional level.
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Part 3, Step 2:
Regulation at
transcription level
Various regulatory
proteins
2
G-C
rich
box
Chromatin
remodeling
3
RNA pol
II
Promoter -proximal
element
mRNA
Promoter
Regulatory region for transcription in eukaryotes
4
Action
Audio Narration
Chromatin remodelling, which provides accessibility to
PLEASE RE_DRAW ALL FIGURES. First
the gene, is a prerequisite for gene expression and is
show the star with text on top followed by
one of the points of regulation at the transcriptional
the arrow pointing to ‘chromatin
level. Remodelling involves acetylation of histone
remodeling’. The grey figure on top must
proteins and demethylation of DNA which are carried
be shown followed by the arrow showing
out by various enzymes. Several regulatory proteins
the figure below. The next heading on right
are also involved in the transcription process after
top must appear and the figure on left
chromatin remodelling, which serve as important
bottom must then be zoomed into to show
points of regulation.
the figure on the right.
Source: An introduction to Genetic analysis by Griffiths, 8th edition (ebook)
As shown
in
animation.
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Description of the action
1
Part 3, Step 3:
Post-transcriptional regulation
2
5’ Capping
3
4
Exons
7-methyl guanosine cap is
added to mRNA shortly
after the start of
transcription, provides
recognition by ribosome
and protection from RNase
Action
As shown
in
animation.
5
Addition of
Poly(A) tail
Splicing
AA
A A
Poly(A) tail
addition
Spliceosome
5’cap
Introns
Description of the action
PLEASE RE_DRAW ALL FIGURES. First
show the heading on top followed by the
three arrows and the headings below that.
Then show each figure below these
headings in the ovals appearing one after
the other as shown.
AAAA
Audio Narration
mRNA synthesized from DNA by transcription undergoes several
post-transcriptional modifications to form the mature mRNA which
then undergoes translation to form proteins. These modifications
are also under regulatory control to moderate the amount of
mRNA produced based on the requirement. Addition of a poly (A)
tail promotes export of the mRNA from the nucleus and protects
the mRNA from degradation. mRNA that does not have these
modifications is usually very unstable and will be degraded.
Source: An introduction to Genetic analysis by Griffiths, 8th edition (ebook)
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Part 3, Step 4:
Masked mRNA is stored in
unfertilized sea urchin eggs. These
are translated on receiving an
appropriate signal and later provide
nourishment and to the eggs when
required
2
Translational regulation
3
Signal
AAAA
5’cap
Poly(A) tail
Protein
4
Action
As shown
in
animation.
5
Description of the action
PLEASE RE_DRAW ALL FIGURES.
First show the heading on top
followed by the three arrows and the
headings below that. Then show
each figure below these headings in
the ovals appearing one after the
other as shown.
Audio Narration
In eukaryotes, the mRNA does not get translated
until it receives the appropriate signal, thereby
serving as another point of control. Before
receiving the signal, enzymes required for the
process will not be present and will get
synthesized only after the signal is obtained.
Source: An introduction to Genetic analysis by Griffiths, 8th edition (ebook); Biochemistry by Lehninger, 4th edition (ebook)
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Part 3, Step 5:
Post-translational regulation
Protein
degradation
2
Protein targeting
and transport
Post-translation
processing
3
Inactive
Protein
Active
Protein
4
Action
As shown
in
animation.
5
Description of the action
PLEASE RE_DRAW ALL FIGURES.
First show the protein structure on
the left followed by the arrow and the
structure on the right with all the
labels as shown. Then show the
arrow on the right and elbow arrow
on the left with labels.
Source: Biochemistry by Lehninger, 4th edition (ebook)
Audio Narration
Proteins undergo several post translational
modifications such as phosphorylation,
acetylation, methylation, glycosylation etc. These
processes are essential for formation of the
active, functional protein. Regulation at the posttranslational level can lead to degradation of the
non-functional protein molecules.
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Interactivity option 1:Step No:1
Cells growing in their exponential phase in a medium of glucose are transferred to a medium
containing only lactose for their carbon source. These cells are again found to have a lag phase
before they again start growing rapidly. What could be the possible reason for this?
Transfer of
cells
2
3
Glucose
Lactose
Bacterial cells
Medium containing
only glucose
4
Interacativity Type
Choose the
correct option
5
Medium containing
only lactose
Options
User must be allowed
to choose any one of
the 4 options given in
the next slide.
Boundary/limits
Results
D is the correct answer,. If user
chooses B, it must turn green
with the remark ‘correct
answer’. If user chooses any of
the remaining options, it must
turn red with the remark
‘incorrect answer’.
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2
Interactivity option 1:Step No:1
Cells growing in their exponential phase in a medium of glucose are transferred to a medium
containing only lactose for their carbon source. These cells are again found to have a lag phase
before they again start growing rapidly. What could be the possible reason for this?
A) Due to difference in pH of the two media.
B) Due to changes in cell morphology.
C) Because of modifications in time required for cell growth and divisions.
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D) Due to the time it takes to induce the lac operon which synthesizes b-galactosidase.
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Questionnaire
1. Negative regulation of the lac operon is brought about by
Answers: a) cAMP b) lacI c) CAP d) None of the above
2
2. Gene regulation is most commonly implemented at which of the following stages?
Answers: a) translation b) transcription c) post-translational modification d) protein
transport
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3. Which of the following is the effector molecule for the repressible system?
Answers: a) Activator b) Co-repressor c) Inducer d) Repressor
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4. What is the binding interaction that takes place during negative regulation in an
inducible system?
Answers: a) Inducer-repressor b) Inducer-inactive activator
inactive repressor
d) Co-repressor- activator
c) Co-repressor –
5. The binding site for the effector molecule is?
5
Answers: a) Promoter b) Enhancer c) Operator d) None of the above
Links for further reading
Books:
Biochemistry by A.L.Lehninger et al., 4th edition
An introduction to Genetics by Griffiths, 8th edition
Molecular Biology of the Gene by James Watson, 5th
edition