Transcript with an intron

Gene Expression
Gene expression?
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.
2
Methods of regulation
• Normally slow relative to metabolic control
• Allows metabolism to be changed in response to
environmental factors
• Transcriptional control most common

Sometimes variation in transcription rate not reflected in
enzyme amount
• Translational control also found

No change in mRNA levels but changes in protein
amounts
3
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.
4
5
RNA Synthesis


DNA template: 3’-to-5’
RNA synthesis: 5’-3’; no primer needed
6
Protein Coding Genes

ORF
• long (usually >100 aa)
• “known” proteins  likely

Basal signals
• Transcription, translation

Regulatory signals
• Depend on organism
 Prokaryotes vs Eukaryotes
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Gene structure relevant to
metabolic regulation
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Promoters
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Eukaryotic gene organization
1.
Transcripts begin and
end beyond the coding
region (5’UTR and
3’UTR)
2.
The primary transcript
is processed by:
5’ capping
3’ formation / polyA
splicing
3.
Mature transcripts are
transported to the
cytoplasm for
translation
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Regulation of gene expression
Promoter Gene (red) with an intron (green)
single copy vs. multicopy plasmids
1. DNA replication
Plasmid
2. Transcription
Primary
transcript
3. Posttranscriptional
processing
mRNA degradation
Mature
mRNA
4. Translation
inactive
protein
5. Posttranslational
processing
active
protein
Protein degradation
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Proteins Regulate Gene Expression
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Proposed Model
rRNA,
tRNA etc.
DNA
Pre-RNA
mRNA
Proteins
13
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
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Intron Splicing in Eukaryotes



GU
at 5’ end
of intron
Exons: coding regions
Introns: noncoding regions
Introns are removed by “splicing”
AG
at 3’ end
of intron
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Splicesomes Roles in Spicing out Intron
RNA splicing occurs in small nuclear ribonucleoprotein
particles (snRNPS) in spliceosomes
Spliceosomes:
protein +
small RNAs
(U1-8)
complementar
y to the
splice
junctions
16
Splicesomes Roles in Spicing 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
2
1
1
2
U1, U2 & U 5
recognize
donor and
acceptor
sites
for splicing
specificity
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Mode of gene regulations

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
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Definitions

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
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Genes Can Be Turned On/Off
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Gene regulation (1)
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
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Gene regulation (2)
Condition 43
upregulated
gene expression
1
2
10
19
down regulated
gene expression
3
4
11
12
20 21
22
5
7
8
13 14 15 16
17
23
6
24
25
9
18
26
constitutively
expressed gene
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Modulators of transcription

Modulators:
(1) specificity factors, (2) repressors, (3) activators
1.
Specificity factors:
Alter the specificity of RNA polymerase
Examples: s-factors (s70, s32 )
s70
Standard Housekeeping gene
promoter
s32
Heat shock Heat shock gene
promoter
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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
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Negative regulation (1)
Repressor
RESULT:
Transcription occurs
when the gene is
derepressed
Effector
Example:
lac operon
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Negative regulation (2)
Repressor
Effector (= co-repressor)
Example:
pur-repressor in E. coli;
regulates transcription of
genes involved in
nucleotide metabolism
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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
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Positive regulation (1)
Activator
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Positive regulation (2)
Activator Effector
RNA polymerase
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Operons
a promoter plus a set of adjacent genes whose
gene products function together.
usually contain 2 –6 genes, (up to 20 genes)
these genes are transcribed as a polycistronic
transcript.
 relatively common in prokaryotes
 rare in eukaryotes
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The lactose (lac) operon
Pi

I
Q3
P
Q1
Z
Q2
Y
A
Contains several elements
•
•
•
•
lacZ gene = b-galactosidase
lacY gene = galactosidase permease
lacA gene = thiogalactoside transacetylase
lacI gene = lac repressor
•
•
•
•
Pi = promoter for the lacI gene
P = promoter for lac-operon
O1 = main operator
O2 and O3 = secondary operator sites (pseudo-operators)
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Regulation of the lac operon
Pi
Pi
II
Q3
Q3
PP Q1
Q1
ZZ
Q2
Q2
LacZ
YY
LacY
AA
LacA
Inducer molecules:
lac repressor
Allolactose:
- natural inducer, degradable
IPTG (Isopropylthiogalactoside)
- synthetic inducer, not metabolized,
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