Transcript DNA Replication

Control of Gene
Expression
Steps of gene expression
 Transcription –
DNA is read to
make a mRNA in
the nucleus of our
cells
 Translation –
Reading the
mRNA to make a
protein in the
cytoplasm
Mainly controlled at the level of transcription
Prokaryotic and eukaryotic gene
organization
Prokaryotic
transcriptional
regulatory regions
(promoters and
operators) lie close to
the transcription start
site
Functionally related
genes are frequently
located near each other
These “operons” are
transcribed into a
single mRNA with
internal translation
initiation sites
Prokaryotic Gene Expression
Expression mainly by controlling transcription
Promoter
Cistron1
Cistron2 CistronN Terminator
Transcription
RNA Polymerase
mRNA 5’
3’
1
2
Translation
C
N
N
N
Ribosome, tRNAs,
Protein Factors
C
N
C
1
2
Polypeptides
3
Operons
A cluster of related genes often coding for enzymes in a
metabolic pathway, which are under the control of a single
promoter regulatory region
Genes that work together are located together
 A promoter plus a set of adjacent genes whose gene
products function together.
They are controlled as a unit
They usually contain 2 –6 genes (up to 20 genes)
These genes are transcribed as a polycistronic transcript.
 It is relatively common in prokaryotes
 It is rare in eukaryotes
Operon System
Regulatory elements
of transcription
Structural genes
: DNA that code for a specific polypeptide
(protein)
Promoter
: DNA segment that recognizes RNA
polymerase
Operator
: Element that serves as a binding site for an
inhibitor protein (modulator) that controls
transcription
Repressor
: Protein which binds to a specific DNA sequences
to determine the transcription of a particular gene
Regulatory gene : Gene encode for repressor protein
Regulatory gene:
Organization of operon
Operons
• The Tryptophan Operon (Repressible and attenuation)
Repressor does not bind to operator unless it interacts
with co repressor
Biosynthetic pathways
• The Lactose Operon (Induction and catabolite
repression)
Repressor is bound to operator unless molecule
to be metabolized is present (inducer)
Catabolic pathways
A repressible operon
Inducible Operon
Lactose Operon
• It codes for the enzymes responsible for lactose catabolism
• Within the operon, there are three genes that code for proteins
(structural protein) and an upstream control region including promoter
and a regulatory site called the operator
• Laying outside the operon is the repressor gene, which codes for a
protein (lac repressor) that binds to the operator site and is responsible
for the suppression of the operon by blocking the binding of RNA
polymerase
• Transcribed mRNA may contain information for more than one protein
(a polycistronic mRNA)
• The synthesis of these mRNA is regulated in accordance with the needs
of the cells at any time thus enable the cell to adapt quickly to changing
environmental conditions
The lactose (lac) operon
Pi
I
Q3
P
Q1
Z
Q2
Y
• Contains several elements
–
–
–
–
lacZ gene = β-galactosidase
lacY gene = galactosidase permease
lacA gene = thiogalactoside transacetylase
lacI gene = lac repressor
–
–
–
–
Pi = promoter for the lacI gene
P = promoter for lac-operon
Q1 = main operator
Q2 and Q3 = secondary operator sites (pseudo-operators)
A
Regulation of the lac operon
Pi
I
Q3
P
Q1
Z
Q2
LacZ
lacI repressor
Y
LacY
Inducer molecules→ Allolactose:
- natural inducer, degradable IPTG
(Isopropylthiogalactoside)
- synthetic inducer, not metabolized
A
LacA
The lac operon:
model for gene expression
Includes three protein
synthesis coding region-sometimes called "genes" as
well as region of
chromosome that controls
transcription of genes
Genes for proteins involved
in the catabolism or
breakdown of lactose
When lactose is absent, no
transcription of gene since
no need for these proteins
When lactose is present,
transcription of genes takes
place so proteins are
available to catalyze
breakdown of lactose
Eukaryotic gene
Eukaryotic gene Expression
1.Transcripts begin and end
beyond the coding region
2.The primary transcript is
processed by:
5’ capping
3’ formation / polyA
splicing
3.Mature transcripts are
transported to the
cytoplasm for translation
Control of Gene Expression
Regulation of gene expression
 Gene expression is regulated—not all genes are constantly
active and having their protein produced
 The regulation or feedback on gene expression is how the
cell’s metabolism is controlled.
 This regulation can happen in different ways:
1. Transcriptional control (in nucleus):
e.g. chromatin density and transcription factors
2. Posttranscriptional control (nucleus)
e.g. mRNA processing
3. Translational control (cytoplasm)
e.g. Differential ability of mRNA to bind ribosomes
4. Posttranslational control (cytoplasm)
e.g. changes to the protein to make it functional
– Regulatory proteins that bind to control sequences
– Transcription factors promote RNA polymerase binding
to the promoter
– Activator proteins bind to DNA enhancers and interact
with other transcription factors
– Silencers are repressors that inhibit transcription
– Control sequences
– Promoter
– Enhancer
– Related genes located on different chromosomes can
be controlled by similar enhancer sequences
Enhancers
Promoter
DNA
Activator
proteins
Transcription
factors
Other
proteins
RNA polymerase
Bending
of DNA
Transcription
Gene
Transcription control
• Transcription factors
• Proximal activators
• Distal control elements
(enhancers)
– DNA binding domain
– Activation domains bind to
other proteins
– These are cell-specific
– A few common structures,
but found in different
combinations in different cells
Eukaryotic gene expression
Gene regulation of the transcription
Condition 2
1
Chr. I
1
10
Chr. II
Chr. III
2
19
“turned “turned
“turned
off”
off”
on”
on”
4
5
6
7
8
3
11
12
20 21
22
constitutively
expressed gene
13 14 15 16
23
induced
gene
24
9
17
25
18
26
repressed
gene
inducible/ repressible genes
Gene regulation
upregulated
gene expression
1
2
10
19
Condition 43
down regulated
gene expression
3
4
11
12
20 21
22
constitutively
expressed gene
5
7
8
13 14 15 16
17
23
6
24
25
9
18
26
Post-Transcriptional Modification in Eukaryotes
 Primary transcript formed first
 Then processed (3 steps) to form mature mRNA
 Then transported to cytoplasm
Step 1: 7- methyl-guanosine “5’-cap”
added to 5’ end
Step 2: introns spliced out; exons link
up
Step 3: Poly-A tail added to 3’ end
mature mRNA
5’-cap- exons -3’ PolyA tail
Alternative picture: co-transcriptional
pre-mRNA processing
Cap Functions
The attachment of 7Me-GTP to the 5’ end of a
nascent mRNA with a 5’ to 5’ phospho-ester
linkage
1.
2.
3.
4.
Protection of the mRNA from degradation
(Protection from 5 exoribonucleases)
Enhances translation in the cytoplasm (Enhancement
of the mRNA’s translatability)
Enhances transport from the nucleus
Proper splicing of the pre-mRNA (Enhances splicing
of the first intron (for some pre-mRNAs))
Intron Splicing
Step by step removal of pre-mRNA and joining of remaining
exons
Removing intron from pre-mRNA
• Exons : coding regions
• Introns : noncoding regions
Polyadenylation
The process of adding poly(A) to RNA
Synthesis of the poly (A) tail involves cleavage
of its 3’end and then the addition of about
40-200 adenine residues to form a poly (A)
tail
Function - Poly(A) enhances both the lifetime
and translatability of mRNA
End Product
 The end products of protein synthesis is a primary
structure of a protein.
 A sequence of amino acid bonded together by peptide
bonds.
aa2
aa1
aa3
aa4
aa5
aa199
aa200
Polyribosome
Groups of ribosomes reading same mRNA simultaneously
producing many proteins (polypeptides).
incoming
large
subunit
1
incoming
small subunit
2
3
4
polypeptide
5
6
7
mRNA
TYPES OF PROTEINS
Enzymes (Helicase)
Carrier (Haemoglobine)
Immunoglobulin (Antibodies)
Hormones (Steroids)
Structural (Muscle)
Ionic (K+,Na+)
Coupled transcription and translation in bacteria
original
base triplet
in a DNA
strand
As DNA is replicated, proofreading
enzymes detect the mistake and
make a substitution for it:
a base
substitution
within the
triplet (red)
POSSIBLE OUTCOMES:
OR
One DNA molecule
carries the original,
unmutated sequence
VALINE
PROLINE
The other DNA
molecule carries
a gene mutation
THREONINE
VALINE
LEUCINE
HISTIDINE
GLUTAMATE
A summary of transcription and translation in a eukaryotic cell