Transcript How to Study DNA 1. Genetic material 2. Expression product What is gene expression? The activation of a gene that results in a.

How to Study DNA
1. Genetic material
2. Expression product
What is gene expression?
The activation of a gene that results in a protein.
Biological processes, such as transcription, and in
case of proteins, also translation, that yield a gene
product.
 A gene is expressed when its biological product is
present and active.
 Gene expression is regulated at multiple levels.
Expression of Genetic Information
Production of proteins requires two steps:
Transcription involves an enzyme (RNA polymerase)
making an RNA copy of part of one DNA strand.
There are four main classes of RNA:
i. Messenger RNAs (mRNA), which specify the amino acid
sequence of a protein by using codons of the genetic code.
ii. Transfer RNAs (tRNA).
iii. Ribosomal RNAs (rRNA).
iv. Small nuclear RNAs (snRNA), found only in eukaryotes.
 Translation converts the information in mRNA into the
amino acid sequence of a protein using ribosomes, large
complexes of rRNAs and proteins.
Expression of Genetic Information
 Only some of the genes in a cell are active at any given
time, and activity also varies by tissue type and
developmental stage.
 Regulation of gene expression is not completely
understood, but it has been shown to involve an array
of controlling signals.
a. Jacob and Monod (1961) proposed the operon model to
explain prokaryotic gene regulation, showing that a genetic
switch is used to control production of the enzymes needed
to metabolize lactose. Similar systems control many genes in
bacteria and their viruses.
b. Genetic switches used in eukaryotes are different and more
complex, with much remaining to be learned about their
function.
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
Three (3) 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
Promoter Region on DNA
 Upstream from transcription start site
 Initial binding site of RNA polymerase and initiation factors (IFs)
 Promoter recognition: a prerequisite for initiation
Prokaryotic promoter regions
-35 site = TTGACA
-10 site: “TATA” box
7
Promoter Region on DNA
(TATA box)
Pol II Eukaryotic
Promoter Elements
Exon Intron Exon
GC box
~200 bp
CCAAT box
~100 bp
TATA box
~30 bp
Gene
Transcription
start site (TSS)
Pol II Eukaryotic
Promoter Elements
Cap Region/Signal
n C A G T n G
TATA box (~ 25 bp upstream)
T A T A A A n G C C C
CCAAT box (~100 bp upstream)
T A G C C A A T G
GC box (~200 bp upstream)
A T A G G C G nGA
General modulators of
transcription
 Modulators:
(1) specificity factors, (2) repressors, (3) activators
1. Specificity factors:
Alter the specificity of RNA polymerase
s70
Standard
promoter
s32
Housekeeping gene
Heat shock
promoter
Heat shock gene
Modulators of transcription
2. Repressors:





mediate negative gene regulation
may impede access of RNA polymerase to the promoter
actively block transcription
bind to specific “operator” sequences (repressor binding sites)
Repressor binding is modulated by specific effectors
Effector
(e.g. endproduct)
Repressor
Operator
Promoter
Coding sequence
Negative regulation
Repressor
Effector
Example:
lac operon
RESULT:
Transcription occurs
when the gene is
derepressed
Negative regulation
Repressor
Effector (= co-repressor)
Example:
pur-repressor in E. coli;
regulates transcription of
genes involved in nucleotide
metabolism
Modulators of transcription
3. Activators:
 mediate positive gene regulation
 bind to specific regulatory DNA sequences (e.g. enhancers)
 enhance the RNA polymerase -promoter interaction and actively
stimulate transcription
 common in eukaryotes
Activator
RNA pol.
promoter
Coding sequence
Positive regulation
Activator
RNA polymerase
Positive regulation
Activator
Effector
RNA polymerase
Prokaryotic 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
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
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
Regulation of gene expression
Promoter
1. DNA replication
Gene (red) with an intron (green)
Plasmid
single copy vs. multicopy plasmids
2. Transcription
3. Posttranscriptional
processing
4. Translation
5. Posttranslational
processing
Primary
transcript
mRNA degradation
Mature
mRNA
inactive
protein
active
protein
Protein degradation
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
 When regulation of gene expression goes wrong—cancer!
Transcription
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
Definitions
Constitutively expressed genes
Genes that are actively transcribed (and translated) under all
experimental conditions, at essentially all developmental stages, or in
virtually all cells.
Inducible genes
Genes that are transcribed and translated at higher levels in response
to an inducing factor
Repressible genes
Genes whose transcription and translation decreases in response to a
repressing signal
Housekeeping genes
–genes for enzymes of central metabolic pathways (e.g. TCA cycle)
–these genes are constitutively expressed
–the level of gene expression may vary
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
Intron Splicing in Eukaryotes
• Exons : coding regions
• Introns : noncoding regions
• Introns are removed by “splicing”
GU at 5’ end
of intron
AG at 3’ end
of intron
36
Splicesomes Roles in Splicing out Intron
RNA splicing occurs in small nuclear ribonucleoprotein
particles (snRNPS) in spliceosomes
37
Splicesomes Roles in Splicing out Intron
 5’ exon then moves to the 3’ splice acceptor site where a
second cut is made by the spliceosome
 Exon termini are joined and sealed
1
2
1
2
1
2
38
Translation
Three parts:
1. Initiation: start codon (AUG)
2. Elongation:
3. Termination: stop codon (UAG)
Translation
Large
subunit
P
Site
A
Site
mRNA
A U G
Small subunit
C U A C U U C G
Initiation
aa1
aa2
2-tRNA
1-tRNA
anticodon
hydrogen
bonds
U A C
A U G
codon
G A U
C U A C U U C G A
mRNA
peptide bond
aa3
aa1
aa2
3-tRNA
1-tRNA
anticodon
hydrogen
bonds
U A C
A U G
codon
2-tRNA
G A A
G A U
C U A C U U C G A
mRNA
aa1
peptide bond
aa3
aa2
1-tRNA
3-tRNA
U A C
(leaves)
2-tRNA
A U G
G A A
G A U
C U A C U U C G A
mRNA
Ribosomes move over one codon
aa1
peptide bonds
aa4
aa2
aa3
4-tRNA
2-tRNA
A U G
3-tRNA
G C U
G A U G A A
C U A C U U C G A A C U
mRNA
aa1
peptide bonds
aa4
aa2
aa3
2-tRNA
4-tRNA
G A U
(leaves)
3-tRNA
A U G
G C U
G A A
C U A C U U C G A A C U
mRNA
Ribosomes move over one codon
aa1
peptide bonds
aa5
aa2
aa3
aa4
5-tRNA
U G A
3-tRNA
4-tRNA
G A A G C U
G C U A C U U C G A A C U
mRNA
peptide bonds
aa1
aa5
aa2
aa3
aa4
5-tRNA
U G A
3-tRNA
G A A
4-tRNA
G C U
G C U A C U U C G A A C U
mRNA
Ribosomes move over one codon
aa4
aa5
Termination
aa199
aa3 primary
structure
of
a
protein
aa2
aa200
aa1
200-tRNA
A C U
mRNA
terminator
or stop
codon
C A U G U U U A G
Translation
Ribosome
Amino Acids forming
Peptide chain
P Site
Met
His
Tyr
A Site
Val
Pro
3’
E Site
tRNA
anti-codon
5’
codon
AUG
CAU
GGA
UAC
GUA
CCU
mRNA strand
Translation
The difference
• Eukaryotic and prokaryotic translation can react differently to
certain antibiotics
Puromycin
an analog tRNA and a general inhibitor of protein synthesis
 Cycloheximide
only inhibits protein synthesis by eukaryotic ribosomes
 Chloramphenicol, Tetracycline, Streptomycin
inhibit protein synthesis by prokaryotic ribosome
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
Prokaryotes vs eukaryotes:
key points
Prokaryotes
Eukaryotes
Operons
(functional grouping)
Monocistronic RNAs
(One mRNA, one protein)
Polycistronic mRNAs
(single mRNA, multiple ORFs)
Ribosome scanning
No splicing
Regulatory sequences lie
near (~100 bp) the start site
Translation is concurrent
with transcription
Often spliced
Regulatory sequences can be
far (>1 kb) from the start site
RNA processing is concurrent
with transcription; translation
occurs in a separate
compartment
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