2. Topics
1. Overview of gene expression
i. Operons
ii. The transcription process
iii. The translation process
2. Regulation of transcription
i. Induction and transcription
ii. Positive controls
iii. Attenuation
i. Global control
3. Catabolite repression
3. 1.Overview of gene expression
Operons
Transcription process
Translation
4. Gene expression
The processes that cells and viruses use to regulate
the way that the information in genes is turned
into gene products.
Gene regulation is essential for viruses, prokaryotes
and eukaryotes .
It increases the versatility and adaptability of
an organism by allowing the cell to express protein
when needed.
5. Switching Genes On & Off
All the cells in our body contain identical set of genes
How do cells become so different?
Express different subsets of genes
Each cell switches genes on or off depending on current need
Needs to be strictly controlled
expression of a gene in a wrong amount, wrong time or wrong cell
type can lead to deleterious phenotype (cell death, cancer)
6. Gene control in eukaryotes
Gene expression in eukaryotes is controlled by a
variety of mechanisms:
those that prevent transcription
those that prevent expression after the protein has
been produced.
7. Regulation Occurs at Many Levels
1. Transcriptional control
2. Post-transcriptional control
3. Transport to cytoplasm
4. mRNA stability
5. Translational control
6. Post-translational control
8. Gene Control in Prokaryotes
Prokaryotes have two levels of gene control.
Transcriptional mechanisms control the synthesis
of mRNA and
translational mechanisms control the synthesis of
protein after mRNA has been produced.
9. Operons
Operons are groups of genes that function to produce
proteins needed by the cell.
Two (2) kinds:
1. Structural genes - code for proteins needed for the
normal operation of the cell.
For example, they may be proteins needed for the breakdown of sugars.
The structural genes are grouped together and a single mRNA molecule
is produced during their transcription.
2. Regulator genes - code for proteins that regulate
other genes.
Operons have not been found in eukaryotes
11. Transcription
DNA is transcribed to make RNA (mRNA, tRNA, and
rRNA)
Transcription begins when RNA polymerase binds to
the promoter sequence
Transcription proceeds in the 5' 3' direction
Transcription stops when it reaches the
terminator sequence
15. Translation
mRNA is translated in
codons (three nucleotides)
Translation of mRNA begins
at the start codon: AUG
Translation ends at
nonsense codons: UAA,
UAG, UGA
Figure 8.2
16. The Genetic Code
64 sense codons on mRNA
encode the 20 amino acids
The genetic code is
degenerate
tRNA carries the
complementary anticodon
Figure 8.2
17. The Process of Translation
Components needed to
begin translations come
together.
Figure 8.9
18. The Process of Translation
On the assembled
ribosome, at tRNA
carrying the first amino
acid in paired with the
start codon on the mRNA.
The place where this firsts
tRNA sits is called the p
site. A tRNA carrying the
second amino acid
approaches.
Figure 8.9
19. The Process of Translation
The second codon of the
mRNA pairs with a tRNA
carrying the second amino
acids joins to the seconds
by a peptide bond. This
attaches the polypeptide
to the tRNA in the p site.
Figure 8.9
20. The Process of Translation
The ribosome moves along
the mRNA until the
second tRNA is in the p
site. The next codon to be
translated is brought into
the a site. The firsts tRNA
now occupies the e site.
Figure 8.9
21. The Process of Translation
The second amino acid is
paired with the start
codon on the mRNA. Is
release from the e site.
Figure 8.9
22. The Process of Translation
The ribosome continues
to move along the mRNA
and new amino acids are
added to the polypeptide
Figure 8.9
23. The Process of Translation
When the ribosome
reaches a stop codon, the
polypeptide is released.
Figure 8.9
24. The Process of Translation
Finally, the last tRNA is
released ,and the
ribosome comes apart.
The released polypeptide
forms a new protein.
Figure 8.9
25.
26. Regulation
Topics:
Induction :
i. Induction: lac operon and
ii. repression: trp operon
Positive and negative controls
Attenuation
Global controls
27. Induction and repression
Constitutive genes are expressed at a fixed rate
Other genes are expressed only as needed such as:
Inducible genes
Repressible genes
28. Enzyme induction
Metabolites or substrates can turn on inactive genes
so that they are transcribed.
In the process of enzyme induction, the substrate, or
a compound structurally similar to the
substrate, evokes the formation of enzyme(s) which
are usually involved in the degradation of the
substrate.
29. Inducible enzymes: Enzymes that are synthesized as
a result of genes being turned on.
Inducer: The substance that activates gene
transcription.
Inducible enzymes are produced only in response to
the presence of a their substrate (produced only
when needed).
So that energy is not wasted to synthesize unneeded
enzymes.
30. Induction of lac operons
The best case of enzyme induction involves the enzymes of
lactose degradation in E. coli.
Only in the presence of lactose does the bacterium synthesize
the enzymes that are necessary to utilize lactose as a carbon
and energy source for growth.
Two enzymes are required for the initial breakdown of
lactose:
lactose permease, which actively transports the sugar into the
cell, and
beta galactosidase, which splits lactose into glucose plus
galactose.
The genes for these enzymes are contained within the lactose
operon (lac operon) in the bacterial chromosome
31. Mechanism of lac operon
The lac operon is an example of an inducible operon
because the structural genes are normally inactive.
They are activated when lactose is present.
32. The region of DNA where the repressor protein binds is
the operator site.
The promoter site is a region of DNA where RNA
polymerase can bind.
The entire unit (promoter, operator, and genes) is
an operon.
The operator acts like a switch that can turn several
genes on or off at the same time.
33. In order for E.coli to digest lactose, it requires three
types of enzymes A, B and C. Hence it requires gene A, B
and C. These are the structural genes.
In normal condition, the genes do not function because
a repressor protein is active and bound to the DNA
preventing transcription.
34. When lactose is present, it acts as an inducer by binding
to the repressor protein, thus preventing it from attaching
to the operator.
RNA polymerase can then bind to the operator and
transcribe the mRNA molecule.
Three different proteins are synthesized.
35. When all of the lactose in the cell has been
catabolized, the repressor protein binds to the operator
and shuts down the operon.
The repressor protein is produced by a regulator gene.
36. Functional and regulatory
components of the lac operon
Lac I Regulatory gene that encodes for the lac Repressor protein that is concerned with regulating the synthesis of the
structural genes in the operon. Lac I is adjacent to the Promoter site of the operon. An active repressor binds to a
specific nucleotide sequence in the operator region and thereby blocks binding of RNAp to the promoter to initiate
transcription. The lac repressor is inactivated by lactose, and is active in the absence of lactose.
O Operator specific nucleotide sequence on DNA to which an active Repressor binds.
P Promoter specific nucleotide sequence on DNA to which RNA polymerase binds to initiate transcription. (The
promoter site of the lac operon is further divided into two regions, an upstream region called the CAP site, and a
downstream region consisting of the RNAp interaction site. The CAP site is involved in catabolite repression of the lac
operon.). If the Repressor protein binds to the operator, RNAp is prevented from binding with the promoter and
initiating transcription. Under these conditions the enzymes concerned with lactose utilization are not synthesized.
Lac Z, Y and A Structural Genes in the lac operon. Lac Z encodes for Beta-galactosidase; Lac Y encodes the lactose permease; Lac A
encodes a transacetylase whose function is not known.
lac lactose, the inducer molecule. When lactose binds to the Repressor protein, the Repressor is inactivated; the operon is
depressed; the transcription of the genes for lactose utilization occurs.
37. Repression
It is the regulatory mechanism that inhibits gene
expression and decrease the synthesis of enzymes
Usually a response to the overabundance of an end-
product of a metabolic pathway
Repression is mediated by regulatory proteins called
repressors
Repressors block the ability of RNA polymerase to
initiate transcription from the repressed genes
38. Repressive operon
Repressible operons are the opposite of inducible
operons.
Transcription occurs continuously and the repressor
protein must be activated to stop transcription.
Example is tryptophan.
It is an amino acids required by an E.coli
39. Repressible operon (trp operon)
•Genes that code for proteins that produce tryptophan are
continuously transcribed.
Figure 8.13
40. If tryptophan is present in the environment E. coli does
not need to synthesize it and the tryptophan-synthesizing
genes should be turned off.
This occurs when tryptophan binds with the repressor
protein, activating it.
41. Unlike the repressor discussed with the lac operon, this
repressor will not bind to the DNA unless it is activated
by binding with tryptophan.
Tryptophan is therefore a co-repressor.
42. The trp operon encodes the genes for the synthesis of
tryptophan.
This cluster of genes regulated by a repressor that binds
to the operator sequences.
The activity of the trp repressor for binding the operator
region is enhanced when it binds tryptophan known as a
corepressor.
Since the activity of the trp repressor is enhanced in the
presence of tryptophan, the rate of expression of
the trp operon is graded in response to the level of
tryptophan in the cell.
Expression of the trp operon is also regulated
by attenuation.
43. Structural Repressor
Genes
Inducible Inactive Active (inhibits)
Operons
Repressible Active Inactive (inhibits
Operons when activated)
44. Tryptophan in negative feedback inhibition
Tryptophan can inhibit the first enzyme in the synthesis
pathway.
The presence of high levels of tryptophan inhibits the
activity of the enzyme as shown in the biosynthesis
pathway below.
45. Positive & negative controls
Most gene expression is controlled at the level of
transcription
Regulation of gene expression by proteins can be either
positive or negative.
Regulation in prokaryotes is usually negative while it is
positive in eukaryotes.
46. The trp and lac operons discussed above are examples of
negative control because a repressor blocks transcription.
In one case (lac operon) the repressor is active and
prevents transcription.
In the other case (trp) the repressor is inactive and must
be activated to prevent transcription.
47. Structural genes Repressor or
regulator
Negative control Inducible operon Inactive Active (inhibits)
(an active repressor
inhibits Repressible operon Active Inactive (inhibits
transcription) when activated)
Positive control (an active regulator Inactive Inactive (promotes
promotes transcription) when activated)
48. Positive control mechanisms require the presence of an
activator protein before RNA polymerase will attach.
The activator protein itself must be bound to an inducer
molecule before it attaches to mRNA.
49.
50. Attenuation
The attenuator plays an important regulatory role
in prokaryotic cells because of the absence of
the nucleus in prokaryotic organisms.
The attenuator refers to a specific regulatory sequence
that, when transcribed into RNA, forms hairpin structures
to stop transcription when certain conditions are not met
51.
52. CATABOLITE REPRESSION
Many inducible operons are not only controlled by their
respective inducers and regulatory genes, but they are
also controlled by the level of glucose in the
environment.
The ability of glucose to control the expression of a
number of different inducible operons is called
CATABOLITE REPRESSION.
53. When levels of glucose (a catabolite) in the cell are high, a
molecule called cyclic AMP is inhibited from forming.
But when glucose levels drop, ATP phosphates are released
until at last forming cAMP:
ATP --> ADP + Pi --> AMP + Pi --> cAMP
cAMP binds to a protein called CAP (catabolite activator
protein), which is then activated to bind to the CAP binding
site.
This activates transcription, perhaps by increasing the affinity
of the site for RNA polymerase. This phenomenon is
called catabolite repression.
55. Catabolite Repression
(a) Growth on glucose or lactose alone (b) Growth on glucose and lactose
combined
During lag time, intracellular cyclic AMP increases, the lac operon
is transcribed, more lactose is transported into the cell. Figure 8.14
