1. regulation of gene expression dr. ishtiaq ahmad khan dr. panjwani center for molecular medicine...
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Regulation of Gene ExpressionRegulation of Gene Expression
Dr. Ishtiaq Ahmad KhanDr. Ishtiaq Ahmad Khan
Dr. Panjwani Center for Molecular Medicine Dr. Panjwani Center for Molecular Medicine and Drug Researchand 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: -factors (TBPs
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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
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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 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
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 co-expression of functionally related proteins.
19Blumenthal 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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