regulation of gene expression dr. ishtiaq ahmad khan
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Regulation of Gene Expression Dr. Ishtiaq Ahmad Khan. Today’s lecture. Gene expression Constitutive, inducible, repressible genes Specificity factors, activators, repressors Negative and positive gene regulation Lac operon Helix-turn-helix motifs Zinc-fingers Leucine zippers. - PowerPoint PPT PresentationTRANSCRIPT
Regulation of Gene Expression
Dr. Ishtiaq Ahmad Khan
Today’s lecture• Gene expression
• Constitutive, inducible, repressible genes
• Specificity factors, activators, repressors
• Negative and positive gene regulation
• Lac operon
• Helix-turn-helix motifs
• Zinc-fingers
• Leucine zippers
What is 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.
Regulation of gene expression
Plasmid
Gene (red) with an intron (green)Promoter
2. Transcription
Primary transcript
1. DNA replication
3. Posttranscriptional processing
4. Translation
mRNA degradation
Mature mRNA
5. Posttranslational processing
Protein degradationinactiveprotein
activeprotein
single copy vs. multicopy plasmids
Gene regulation (1)
Chr. I
Chr. II
Chr. III
Condition 1
“turned on”
“turned off”
Condition 2
“turned off”
“turned on”
1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25 26
constitutively expressed gene
induced gene
repressedgene
inducible/ repressible genes
Gene regulation (2)
constitutively expressed gene
1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25 26
Condition 3 Condition 4 upregulated gene expression
down regulated gene expression
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
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
Modulators of transcription• Modulators:
(1) specificity factors, (2) repressors, (3) activators
1. Specificity factors:Alter the specificity of RNA polymerase
Examples: -factors (TBPs
70 32
Heat shock geneHousekeeping gene Heat shock promoter
Standard promoter
Modulators of transcription2. Repressors:
mediate negative gene regulationmay impede access of RNA polymerase to the
promoteractively block transcriptionbind to specific “operator” sequences (repressor
binding sites) Repressor binding is modulated by specific effectors
Coding sequence
Repressor
Operator
Promoter
Effector(e.g. endproduct)
Negative regulation (1)
Source: Lehninger pg. 1076
Repressor
EffectorExample: lac operon
RESULT:Transcription occurs when the gene is derepressed
Negative regulation (2)
Source: Lehninger pg. 1076
Repressor
Effector (= co-repressor)Example: pur-repressor in E. coli; regulates transcription of genes involved in nucleotide metabolism
Modulators of transcription3. 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
Coding sequence
Activator
promoter
RNA pol.
Positive regulation (1)
Source: Lehninger pg. 1076
RNA polymerase
Activator
Positive regulation (2)
Source: Lehninger pg. 1076
RNA polymerase
Activator Effector
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
The lactose (lac) operon
• Contains several elements– lacZ gene = -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)
Pi P Z Y A I O3 O1 O2
The lac operon consists of three structural genes, and a promoter, a terminator,regulator, and an operator. The three structural genes are: lacZ, lacY, and lacA.
• lacZ encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose
into glucose and galactose.• lacY encodes β-galactoside permease (LacY),
a membrane-bound transport protein that pumps lactose into the cell.
• lacA encodes β-galactoside transacetylase (LacA), an enzyme that transfers an acetyl group from acetyl-CoA to β-galactosides.
• Only lacZ and lacY appear to be necessary for lactose catabolism.
Theodor Hanekamp © 2003 18
First Level• The lacI gene coding for the repressor lies nearby the lac operon
and is always expressed (constitutive).• Hinder production of β-galactosidase in the absence of lactose. • If lactose is missing from the growth medium, the repressor binds
very tightly to a short DNA sequence called the lac operator. • The repressor binding to the operator interferes with binding of RNA
Pol to the promoter, and therefore mRNA encoding LacZ and LacY is only made at very low levels.
• When cells are grown in the presence of lactose, however, a lactose metabolite called allolactose , which is a combination of glucose and galactose, binds to the repressor, causing a change in its shape.
• Thus altered, the repressor is unable to bind to the operator, allowing RNAP to transcribe the lac genes and thereby leading to higher levels of the encoded proteins.
19
Second Level• The second control mechanism is a response to glucose, which
uses the Catabolite activator protein (CAP) to greatly increase production of β-galactosidase in the absence of glucose.
• Cyclic adenosine monophosphate (cAMP) is a signal molecule whose prevalence is inversely proportional to that of glucose.
• It binds to the CAP, which in turn allows the CAP to bind to the CAP binding site (a 16 bp DNA sequence upstream of the promoter on the left in the diagram below),
• which assists the RNAP in binding to the DNA. In the absence of glucose, the cAMP concentration is high and binding of CAP-cAMP to the DNA significantly increases the production of β-galactosidase
• enabling the cell to hydrolyse (digest) lactose and release galactose and glucose.
Theodor Hanekamp © 2003 20
Theodor Hanekamp © 2003 21
Regulation of the lac operon
Pi P Z Y A I Q3 Q1 Q2
Inducer molecules: Allolactose: - natural inducer, degradableIPTG (Isopropylthiogalactoside)- synthetic inducer, not metabolized,
lacI repressor
Pi P Z Y A I Q3 Q1 Q2
LacZ LacY LacA
Selected DNA binding motifs1. Helix-turn-helix
• Homeodomain
2. Zinc Fingers• Cys4 zinc finger• Cys2 His2 zinc finger (e.g. TFIIIA)
3. Basic domains• Leucine zippers factors (bZIP)• Basic helix-loop-helix (bHLH)
4. Beta-scaffold factors with minor groove contacts
• HMG (High mobility group) proteins
Helix-turn-helix motifs
GENEGalR A T I K D V A R L A G V S V A T V S R V I N-cro F G Q T K T A K D L G V Y Q S A I N K A I HP22-cro G T Q R A V A K A L G I S D A A V S Q W K E
Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
alpha - helix turn alpha - helix
Structure:• about 20 amino acids long• 2 short alpha helicies ( 7 – 9 amino acids long)• DNA recognition helix (binds specific DNA sequence)• Recognition helix and 2nd helix form ~ 90° angle• very short turn ( NOT a beta-turn) • Often glycine at start of the turn (helix breaker)
How does the lac repressor bind DNA?
Source: Lehninger pg. 1082
DNA
DNA recognition helix
LacI repressor (helix-turn-helix domain)
turn
Second alpha helix
Zinc-Finger Motifs
C
C
H
HZn
C
C
H
HZn
C
C
H
HZn
Several subtypes (Cys4, Cys2-His2 …)• Example: Cys2 His2 type• Zinc does not interact with DNA• Usually multiple zinc-fingers in a row • At least some also bind RNA• Consensus sequence:
[Y,F]-X-C-X2-4-C-XXX-F-XXXXX-L-XX-H-X3-5-H
Basic domainsLeucine zippers (bZip):• Basic region of the protein binds to DNA• Mainly act as dimers or other sometimes as other multimers• Special alpha-helices allow formation of coiled-coil
structures.• Hydrophobic residues (Leu) align on one side of the helix• Example: Jun and Fos
7 7 7 7
Source: Lehninger pg. 1084
abcdefg
Leucine zippers
DNA
Leucines
Source: Lehninger pg. 1084
Transcription attenuation
29
Some Genes Are Regulatedby Genetic Recombination
30Example of Salmonella typhimurium
Regulation of Eukaryotic Gene Expression
Gene Regulation at DNA Level
Chromatin Remodeling
1. Changes of DNA Topo structure
Formation of ssDNA
DNase I hypersensitive site
DNA Methylation
2. DNA Methylation
CpG islands
----- are genomic regions that contain a high
frequency of CG dinucleotides.
----- CpG islands particularly occur at or near
the transcription start site of housekeeping genes.
Active transcriptionUnmethylated CpG island
TF RNA pol
Repressed transcription
Methylated CpG island
TF RNA pol
CH3 CH3 CH3
3. Histone modification
methylation
acetylation
TFTF
37
Functions of Histone methylation in transcription
Most well-studied histone modifications are involved in control of transcription.Actively transcribed genesTwo histone modifications are particularly associated with active transcription:•Trimethylation of H3 lysine 4 (H3K4Me3) at the promotor of active genes •Trimethylation of H3 lysine 36 (H3K36Me3) in the body of active genesRepressed genesThree histone modifications are particularly associated with repressed genes:•Trimethylation of H3 lysine 27 (H3K27Me3)•Di and tri-methylation of H3 lysine 9 (H3K9Me2/3)•Trimethylation of H4 lysine 20 (H4K20Me3)
38
Acetylated histones and nucleosomes represent a type of epigenetic tag within chromatin. Acetylation removes the positive charge on the histones, thereby decreasing the interaction of the N termini of histones with the negatively charged phosphate groups of DNA. As a consequence, the condensed chromatin is transformed into a more relaxed structure that is associated with greater levels of gene transcription.
Functions of Histone methylation in transcription
7.3 Transcriptional Regulation
1. Cis-acting element
(1) What is cis-acting element?
Concept
Cis-acting elements - DNA sequences close
to a gene that are required for gene expression
2. What is trans-acting factor?
Concept
trans-acting factors - usually they are
proteins, that bind to the cis-acting elements to
control gene expression.
These trans-acting factors can control gene
expression in several ways:
may be expressed in a specific tissue
may be expressed at specific time in development
may be required for protein modification
may be activated by ligand binding
Domains of trans-acting factors
DNA binding domain DBD
transcription activating domain
Post-Transcriptional Regulation
1. Gene Regulation of mRNA Processing
exon shuffling
alternative gene splicing
2. Gene Regulation of mRNA Editing
3. mRNA Longevity
4. mRNA Transport Control
5. RNA Interference (RNAi)
miRNA
siRNA
7.5 Translational and Post-translational Regulation
1. Translation Control
Blocking mRNA Attachment to Ribosomes
2. Regulation of Protein Processing
Protein Modification
3. Regulation of Protein Stability