Transcript Dr Ishtiaq Regulation of gene expression

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Regulation of Gene Expression
Dr. Ishtiaq Ahmad Khan
Dr. Panjwani Center for Molecular Medicine
and Drug Research
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: s-factors (s70, s32 ), TBPs
s70
Standard Housekeeping gene
promoter
s32
Heat shock Heat shock gene
promoter
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 (1)
Repressor
RESULT:
Transcription occurs
when the gene is
derepressed
Effector
Example:
lac operon
Source: Lehninger pg. 1076
Negative regulation (2)
Repressor
Effector (= co-repressor)
Example:
pur-repressor in E. coli;
regulates transcription of
genes involved in
nucleotide metabolism
Source: Lehninger pg. 1076
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 (1)
Activator
RNA polymerase
Source: Lehninger pg. 1076
Positive regulation (2)
Activator Effector
RNA polymerase
Source: Lehninger pg. 1076
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
Pi
I
O3
P
O1
Z
O2
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)
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 βgalactosides.
• Only lacZ and lacY appear to be necessary for
lactose catabolism.
Theodor Hanekamp ©
2003
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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.
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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),
• 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.
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Regulation of the lac operon
Pi
I
Q3
P
Q1
Z
Q2
LacZ
Y
LacY
Inducer molecules:
lacI repressor Allolactose:
- natural inducer, degradable
IPTG (Isopropylthiogalactoside)
- synthetic inducer, not metabolized,
A
LacA
Operons in eukaryotes
 Dicistronic transcription units specify a messenger RNA (mRNA)
encoding two separate genes that is transported to the cytoplasm
and translated in that form. Presumably, internal ribosome entry
sites (IRES), or some form of translational re-initiation following the
stop codon, are responsible for allowing translation of the
downstream gene.
 In the other type, the initial transcript is processed by 3΄ end
cleavage and trans-splicing to create monocistronic mRNAs that are
transported to the cytoplasm and translated.
 Like bacterial operons, eukaryotic operons often result in coexpression of functionally related proteins.
Blumenthal T, BRIEFINGS IN FUNCTIONAL GENOMICS AND PROTEOMICS. VOL 3. NO 3. 199–211. NOVEMBER 2004
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Genes whose function is to specify
mitochondrial proteins and those that
encode the basic machinery for gene
expression, transcription, splicing and
translation have a very strong tendency to
be transcribed in operons
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Examples in C. elegans
 An operon that expresses two subunits of the
acetylcholine receptor.
 An operon that encodes two proteins needed
for modifying collagen, expressed only in
collagen-producing cells.
 An operon that co-expresses an ion channel
protein with a protein that modifies the activity of
that channel;
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