defects in gene regulation can alter the development of an organism regulation of gene expression
TRANSCRIPT
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Defects in gene regulation can alter the development of an organism
Regulation of Gene Expression
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Seven processes that affect the steady-state concentration of a protein in a cell
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Regulation of Gene Expression
• Principles of gene regulation
• Regulation of gene expression in prokaryotes
• Regulation of gene expression in eukaryotes
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Principles of Gene Regulation
constitutive vs. regulated gene expression
housekeeping genes, gene products that are required at all times at a more or less constant level.e.g., in citric acid cycle
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Principles of Gene Regulation
1) RNA polymerase binds to DNA at promoters
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Principles of Gene Regulation (cont’d)
2) Transcription initiation is regulated by proteins that bind to or near promoters.
Repression of a repressible gene:(i.e., negative regulation) repressors (vs. activators) bind to operators of DNA.Repressor is regulated by an effector, usually a small molecule or a protein, that binds and causes a conformational change.Activator binds to DNA sites called enhancer to enhance the RNA polymerase activity. (i.e., positive regulation)Induction of an inducible gene, e.g., heat-shock genes.
Heat-shock promoters
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Principles of Gene Regulation (cont’d)
3) Most prokaryotic genes are regulated in units called operons.
Francois Jacob & Jacques Monod, 1960
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Lactose metabolism in E. coli
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4) The lac operon is subject to negative regulation: repressor
tetramericrepressor
uninduced
IPTG induced
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5) Regulatory proteins have discrete DNA-binding domains
Functional groups (pink) in DNA available for protein binding
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5) Regulatory proteins have discrete DNA-binding domains
e.g., specific amino acid-base pair interactions in DNA-protein interaction
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e.g., a DNA-binding domain (3) interacts directly with DNA at major groove
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AATTGT…ACAATTTTAACA…TGTTAA
The DNA binding sites for regulatory proteins are often inverted repeats of a short DNA sequence (a palindrome) at which multiple subunits (usually two) of a regulatory protein bind cooperatively.
e.g., Lac repressor vs. operatorinverted repeats
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Examples of DNA-binding motifs/domains:
• helix-turn-helix: e.g., Lac repressor• zinc finger: e.g., Zif 268• homeodomain: e.g., Ultrabithorax (Ubx)
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Helix-turn-helix
DNA-binding domains
Lac repressor,a tetramer
allolactose-bindingdomains
hydrogen-bonding (red)hydrophobic interactions (yellow)
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• zinc finger:
Zn2+
(2 Cys, 2 His)
~30 a.a.
In many eukaryotic (few prokaryotic) DNA-binding proteins
e.g., Zif 268
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• homeodomain:
homeobox: DNA sequence encoding homeodomain
a helix (red) protruding into the major groove
e.g., Ultrabithorax (Ubx)
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6) Regulatory proteins also have protein-protein interaction domains
Leucine zippers
• Leucine zippers• basic helix-loop-helix
interacting Leu (red)
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basic helix-loop-helixe.g., transcription factor Max (dimeric)
DNA-binding segment (pink)
helix-loop-helix
(red & purple)
A pair of interacting Leu
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Regulation of Gene Expression
• Principles of gene regulation
• Regulation of gene expression in prokaryotes
• Regulation of gene expression in eukaryotes
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The Lac Operon
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The lac Operon Is Subject to Positive Regulation:Activation by CRP (cAMP receptor protein)
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CRP homodimer
cAMP (pink)
DNA is bended
Region interacting with RNA polymerase (yellow)
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The effect of glucose on CRP is mediated by cAMP.Transcription occurs only at low glucose and high lactose.
cAMP & CRP are involved in the coordinated regulation of many operons. A net of operons with a common regulatoris called regulon.
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The ara operonundergoes both positive & negative regulationby a single regulatory protein AraC.
Th end product of the arabinose metabolic pathway,D-xylulose 5-phosphate, is an intermediate in thepentose phosphate pathway.
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When the AraC repressor is depleted, The araC gene is transcribed from its own promoter.
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At high glucose and low arabinose, AraC binds andbrings araO2 and araI sites together to form aDNA loop, repressing araBAD.
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At low glucose, but arabinose is present, AraC repressor binds arabinose and changes conformation to become an activator.DNA loop is opened, and AraC binds to each half-site of araI and araO1. The proteins interact with each other, and act in concert with CRP-cAMP to facilitate transcription of the araBAD genes.
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Many Genes for Amino Acid Biosynthesis Are Regulated by Transcription Attenuatione.g., the trp operon
At high tryptophen, 1) the repressor binds its operator, 2) transcription of trp mRNA is attenuated.
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Trp repressordimeric,helix-turn-helix
bound tryptophen (red)
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The trp mRNA leader (trpL):Sequence 1 encodes a small peptide, leader peptide,containing two Trp residues.
Transcription attenuation in the trp operon
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Transcription attenuation in the trp operonattenuator
At high trp
At low trp
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The Trp Operon
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Induction of the SOS Response in E. coli Requires Destruction of Repressor Protein LexA:
Coprotease RecA is activated by DNA damage (single stranded DNA)LexA is cleaved and inactivated by RecA
Operon-like regulation
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mRNAs of some ribosomal proteins (r-protein):
r-protein acts as a translational repressor
Synthesis of Ribosomal Proteins Is Coordinated with rRNA Synthesis
yellow: RNA pol subunitsblue: EFs
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Synthesis of Ribosomal Proteins Is Coordinated with rRNA Synthesis
e.g., stringent response in E. coli, response to amino acid starvation
uncharged tRNA binding > stingent factor (RelA) binding >catalysing ppGpp synthesis > binding to -subunit of pol >rRNA synthesis reduced
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Some Genes Are Regulated by Genetic Recombination
e.g., regulation of flagellingenes in Salmonella: phase variation allows evasion of host immune response.
repressor
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Regulation of Gene Expression
• Principles of gene regulation
• Regulation of gene expression in prokaryotes
• Regulation of gene expression in eukaryotes
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Extraordinary complexity of gene regulation in eukaryotes
• Transcriptional Active Chromatin is Structurally Different from Inactive Chromatin: hypersensitive sites (100 ~ 200 bp), DNaseI sensitive sequences whithin the 1000 bp flanking the 5’ end of transcribed genes.• Modifications Increase the Accessibility of DNA: e.g., 5’-methylation of cytosine of CpG sequences is common in eukaryotic DNA, active genes tend to be undermethylated.• ……
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Extraordinary complexity of gene regulation in eukaryotes
• Chromatin Is Remodeled by Acetylation and Nucleosome Displacements chromatin remodeling: the detailed mechanisms for transcription-associated structure changes in chromatin.
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Extraordinary complexity of gene regulation in eukaryotes
• Many Eukaryotic Promoters Are Positively Regulated• DNA-Binding Transactivators and Coactivators Facilitate Assembly of the General Transcription Factors enhancer in higher eukaryotes, upstream activator sequences (UASs) in yeast.
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Three Classes of Proteins Are Involved in Transcriptional Activation
basal transcription factors, DNA-binding transactivators, and coactivators.
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A wide variety of repressors function by a range of mechanisms
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The Genes Required for Galactose Metabolism in Yeast Are Subject to Both Positive and Negative Regulation
regulated 6 genes (table 28-3)regulatory proteins:Gal4p, Gal80p & Gal3p
Binding of galactose to Gal3p and its interaction with Gal80p produce a conformation change in Gal80p that allows Gal4p to function in transcription activation.
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Unlike bacteria, there is no operons in yeast. Each of the GAL genes is transcribed separately.
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The GAL system is shown to illustrate the transcription activation of a group of related eukaryotic genes.
The initiation complexes assemble stepwise:1) DNA-binding transactivators2) Basal transcription factors/pol II3) Additional protein complexes needed to remodel the chromatin
e.g., SWI/SNF: histone remodeling SAGA: histone acetylation
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Typical DNA-binding transactivators have a DNA-binding domain and an activation domain.e.g., Gal4p, acidic activation domain function in activation CTF1(CCAAT-binding transcription factor 1), proline-rich activation domain Sp1, glutamine-rich activation domain
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“Domain-swapping” experiment: A chimeric protein containing the DNA-binding domain of Sp1 and the activation domain of CTF1 activates transcriptionif a GC box is present.
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Eukaryotic Gene Expression Can Be Regulated by Intercellular and Intracellular Signalse.g., steroid hormone (estrogen) receptors
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Regulation Can Occur through Phosphorylation of Nuclear Transcription Factors
e.g., The catalytic subunit of protein kinase A, released when cAMP levels rise, enters the nucleus and phosphorylates a nuclear protein, the CRE-binding protein (CREB), >> binding to CREs near certain genes and acting as a transcription factor.
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Many Eukaryotic mRNAs Are Subject to Translational Repression
e.g., translational repressors (RNA-binding proteins) interact with initiation factors or with the ribosome to prevent or slow translation
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Development Is Controlled by Cascades of Regulatory Proteins
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Maternal Genes
Christiane Nusslein-Volhard (p.1112)bicoid (bcd) gene product gradient
Two posteriors
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Segmentation genese.g., ftz gene product (Ftz) Early embryo
Late embryo
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Homeotic genes:
Antennapaedia (~ mouse HOX 1.1)
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bithorax mutation
The discovry of structural determinants with identifiable molecular functionsis the first step in understanding the molecular events underlying development.