340 - 04 -- gene expression
TRANSCRIPT
Differential Gene Expression• Transcriptional control
• RNA processing control
• Translational control
• Post-translational control
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Transcriptional control DNA-mRNA
mRNA processing and stability
Translational control mRNA -Protein
Post-Translational control Protein modifications
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Promoter Transcription initiation site Translation initiation site
• 5’ UTR (untranslatedsequence)
Exons (expressed) Introns (intervening) Translation termination codon 3’UTR
• Polyadenylation addition site
Transcription termination site
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Transcription initiation site
Translation initiation site
Translation termination codon
3’ Untranslated region
Promoters
Exons
Introns
Three main regulatory agents:• Nuclear proteins that regulate access to DNA
• Multiple, alternative promoters
• Cells differ in number or type of transcription
factors
Let’s take a look at each…..
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Nuclear proteins regulate access to DNA
DNA normally bound up in nucleosomes (histone proteins)
Chromatin remodeling proteins unwind specific DNA sequences allowing access to transcription initiation complex
• Exposes enhancer and promoter regions allowing transcription
Detour -- now we need to learn what enhancers & promoters are……
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Chromatin remodeling affects• DNA replication & repair
• Apoptosis
• Chromosome segregation
• Pluropotency
Targeting chromatin remodeling pathways
is a growing field in targeting several
cancers (regulate mitosis)
Promoter -- initiates transcription of a particular gene (TATA box, GC islands)
Enhancer -- short region of DNA which can be found with proteins (TF’s) to “enhance” transcription levels in a gene cluster
Transcription factor -- works in conjunction with enhancers; family of proteins; alter DNA confirmation
Silencer -- “negative” enhancers; DNA sequence which binds repressors preventing RNA polymerase from initiating transcription
Know this
Direct methylation of DNA
Methyltransferases –enzymes that add a methyl group
Common in CpG sequences• “Cytosine – phosphate-Guanine”
In mammals, 70 – 80% of CpG sites are methylated
Repression mechanism:• Blocks transcription factors
Enhancers:• Specific sequence in DNA• Short series of bases• Vary in location• Can be far away from gene regulated • Bound by transcriptional activators (molecules from
inside or outside the cell that aid in transcription)
Silencers• Specific sequence of DNA• Short series of bases• Bind proteins that promote assembly of large protein
complexes that block transcription
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Know qualities
Many enhancers cause DNA looping• Transcriptional activator
binds to enhancer
• Activator also interacts with part of transcription complex to recruit complex to gene by bending DNA
• Complex then interacts with promoter and recruits polymerase holenzyme
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Know this
Enhancers are hard to find because:• Variable in sequence from one gene to the next
• Short series of bases
• Vary in location
• Can be far away from gene regulated
So -- how do you find them?
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“Reporter Constructs”• Put region of interest (with
regulatory elements) upstream
of an inducible gene (such as
lacZ)
• Modify (by deletion or
substitution) presumed
regulatory region
• Modifications that alter
expression of inducible gene
are regulatory elements
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mRNA processing results in same mature mRNA
Alternative promoters allow for different regulation of same gene• Larval vs. adult expression
Fetal gamma-globin to adult beta-globin in humans
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Some genes have more than one promoter sequence
Different promoters produce different primary
transcripts
3 major domains:• DNA binding domain
• Activating domain – interacts with initiation complex proteins
• Protein-protein interaction domain
Major transcription factor families• Homeodomain – Hox genes (axis formation)
• Basic helix-loop-helix – muscle & nerve specification
• Basic leucine zipper – liver & fat cell differentiation
– Zinc Finger - segmentation, secondary sex determination, metamorphosis, limb development
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Know this