Regulation of Gene Expression
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Transcript of Regulation of Gene Expression
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Regulation of Gene Expression• Inducible gene expression
– kinetics of β-galactosidase enzyme induction
– Add inducer• start transcription = mRNA
accumulation
• mRNA translation = protein accumulation
– Remove inducer• Stop transcription (txn)• mRNA and protein levels slowly
decay back to original level
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Gene expression: bacteria• Inducible gene expression example: Sugar catabolism
– In the absence of lactose, no need to have enzymes that metabolize it– In the presence of lactose, cell should make enzymes for metabolizing it
• Repressible gene expression example: Amino acid anabolism– In the absence of tryptophan, cell must synthesize tryptophan– In the presence of tryptophan, cell does not need to make it
• Both systems make use of a Transcriptional (TXN) Repressor protein– DNA binding protein that interferes with TXN
• Acts as an ON/OFF switch for gene expression• Binds a DNA sequence called the “Operator”• Steric blockade to promoter binding
• Binds relevant metabolite that allosterically affects DNA binding
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Gene expression: bacteria• Inducible gene expression example: Sugar catabolism
– In the absence of lactose, no need to have enzymes that metabolize it
Minus Lactose, Lac Repressor binds Operator and blocks TXN
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Gene expression: bacteria• Inducible gene expression
example: Sugar catabolism – In the presence of lactose, cell
should make enzymes for metabolizing it
+ Lactose, Lac Repressor can’t bind Operator
Allosteric effector inactivates Lac Repressor DNA binding
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Gene expression: bacteria• Inducible gene expression
– Lac operon can only be induced when glucose level is low– Low glucose = high cAMP level inside cell– CRP protein binds and activates TXN in presence of cAMP
• cAMP-CRP complex binds DNA• Helps RNAp bind to promoter region
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Gene expression: bacteria• Repressible gene expression
example: Amino acid anabolism– In the absence of tryptophan,
cell must synthesize tryptophan
• Trp Repressor can only bind to Operator sequence when tryptophan is present
Minus Tryptophan, Trp Repressor can’t bind Operator
Allosteric effector needed for effective DNA binding
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Gene expression: bacteria• Repressible gene expression
example: Amino acid anabolism– In the presence of tryptophan,
cell does not need to make it
• Trp Repressor can only bind to Operator sequence when tryptophan is present
+ Tryptophan, Trp Repressor binds Operator tightly, blocks TXN
Allosteric effector needed for effective DNA binding
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Structure of the nucleus• Nucleoli: rDNA, rRNA synthesis, ribosome assembly• Chromatin: Genomic DNA - protein complexes, transcription• Nuclear envelope
– Two membranes (10-50nm separation)
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Structure of the nucleus• Nucleoli: rDNA, rRNA synthesis, ribosome assembly• Chromatin: Genomic DNA - protein complexes, transcription• Nuclear envelope
– Two membranes (10-50nm separation)
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Structure of the nucleus• Nuclear envelope
– Two membranes (10-50nm separation)• Continuous with endoplasmic reticulum (ER)
• Supported on nuclear side by nuclear lamina– Meshwork of proteins on inner surface for mechanical support– Lamins are related to intermediate filaments of cytoskeleton
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Structure of the nucleus• Supported on nuclear side by nuclear lamina
– Meshwork of proteins on inner surface for mechanical support– Lamins are related to intermediate filaments of cytoskeleton
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Hutchinson-Gilford Progeria Syndrome• Caused by mutations in
Lamin A– Premature aging– Most die by age 13
• Molecular phenotype is abnormal nuclei shape
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The Nuclear Pore Complex (NPC)• Gateway into the nucleus
– 15-30 times larger than a ribosome– Composed of ~30 nucleoporin proteins– Molecules with molecular weights
<40,000 can pass through freely
view from cytoplasm
view from nucleus
side viewwith goldparticles
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Molecular weights (MW)• Most ions are very small
– Na+ 23g/mol– Cl- 35g/mol– K+ 39g/mol– Mg2+ 24g/mol– Ca2+ 40g/mol– PO43- 95g/mol
• Amino acids (AAs) sizes – Glycine 75g/mol– Tryptophan 204g/mol– Average ~110g/mol
• Average human protein length– 375 AAs~41,250g/mol
1 kiloDalton (kDa) = 1000g/mol
– 375 AAs~41 kDa
– Most eukaryotic proteins will not freely diffuse through the nuclear pore complex
– Nuclear entry and exit is regulated
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Regulation of nuclear import/export• Proteins contain nuclear import and/or nuclear export signal sequences
– Import: Nuclear Localization Signal (NLS): n-PKKKRKV-c– Importin beta/alpha binds to the NLS of the “cargo” protein in the cytoplasm– The beta-alpha-”cargo” complex binds the cytoplasmic filaments of the NPC– The docked complex translocates through the NPC to the nucleoplasm
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Regulation of nuclear import/export• Proteins contain nuclear import and/or nuclear export signal sequences
– Import: Nuclear Localization Signal (NLS): n-PKKKRKV-c– On nuclear side, Ran-GTP binds and disrupts the beta-alpha-”cargo” complex
• Cargo is released in nucleus• Importin-beta is bound to Ran-GTP
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Regulation of nuclear import/export• Proteins contain nuclear import and/or nuclear export signal sequences
– Ran-GTP bound to Importin-beta travels down its concentration gradient• Cytoplasmic Ran-GTP hydrolyzes its bound GTP• Ran-GDP releases Importin-beta in cytoplasm
– Export: Nuclear Export Signal (NES)• Exportin carries alpha back to cytoplasm
Ran-GTPRan-GDPGTP GDP
Ran-GTPRan-GDP
Pi
GNEF
GAP
(bind beta)
(release beta)
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Gene expression: eukaryotes
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Chromosomes and chromatin• Chromatin = DNA + associated proteins
– Histone octamer• ( H2A, H2B, H3, H4 ) x2
– Nucleosome = histone octamer + 146 bp DNA
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Chromosomes and chromatin• Chromatin = DNA + associated proteins
– H1 linker protein connects adjacent nucleosomes• 10nm “beads-on-a-string” compacts to a 30nm fiber• Packaged DNA is protected from damaging agents• Octamer tails also contribute to higher-order compaction
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Euchromatin & heterochromatin• Euchromatin
– Dispersed, not compacted• Readily accessed by TXN
factors and RNAp• Transcriptionally active
– Histone modifications• Histone Acetyltransferase
enzymes (HATs)• Acetylation of Lysine
residues in H3 and H4
DNA (-) <--> Histones (+)
• Neutralize (+) on histones, reducing DNA - histone tail interaction
• Create binding sites for additional factors Acetyl-lysineLysine
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Euchromatin & heterochromatin• Heterochromatin
– Highly compacted• Not readily accessed by
TXN factors or RNAp• Transcriptionally inactive
– Histone modifications• Histone Methylransferase
enzymes (HMTs)• Methylation of Lysine
residues in H3 and H4• Create binding sites for
additional factors
– Constitutive: always compacted– Facultative: conditionally
compacted (e.g. cell type specific)• X-inactivation in females
trimethyl-lysineLysine+
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X Chromosome inactivation• Males have only 1 X Chromosome• Females have 2 X Chromosomes
– Gene dosage in females is regulated by only using one of the two available X chromosomes
– Cats have a pigment gene on the X chromosome• Black allele (Xb) versus
Orange allele (Xo)• Female calico cats have one Xb
allele and one Xo allele• Random inactivation of Xb or Xo
yields orange or black patches• Cloning of a calico cat confirmed
the random nature of X-inactivation
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X Chromosome inactivation• Convert one X chromosome to facultative heterochromatin• Random event early in development• Stably maintained through subsequent cell divisions
Before inactivation
After
Xb
XoXb
Xo
Xb
XoXb
XoXb
XoXb
Xo
Xb
XoXb
Xo
Xb
Xo
Xb
XoXb
Xo
Xb
XoXb
XoXb
XoXb
Xo
female male
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X Chromosome inactivation• Actively transcribed chromosomes stain
strongly for acetylated histones• Inactivated X chromosome does not• Histones of inactivated X are instead
methylated by a HMT enzyme• HP1 binds methylated sites and facilitates
chromatin condensation
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Gene expression: eukaryotes• TXN-level control
– TXN factors bind specific DNA sequence “elements”• Activators
– DNA binding domain + activation domain
• Repressors– DNA binding domain +
repression domain
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Txn-level control• Promoter structure
– TATA Box (core element)– Response elements < 1 kb away
• Can be isolated sites for individual factors or clustered together– Enhancer elements > 1 kb away
• 200 bp size containing many binding sites– Insulator elements separate one transcription unit from an adjacent unit
ENHANCEINSULATE
PEPCK gene
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Txn-level control• Co-activation
– Co-operative binding between Activator and GTFs
– Histone modification: recruit HAT enzymes
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Txn-level control• Co-activation
– Nucleosome remodeling: recruit chromatin remodeling complexes
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Txn-level control• Co-activation
– Nucleosome remodeling: recruit chromatin remodeling complexes
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Txn-level control• Co-activation
– Cleared promoter region now accessible to TFIID, other GTFs and RNApII
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Txn-level control• Co-repression
– Antagonistic binding: block GTFs
– Histone modification• recruit Histone deacetylase
(HDAC) enzymes
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Txn-level control• Co-repression
– Antagonistic binding: block GTFs
– Histone modification• recruit Histone deacetylase
(HDAC) enzymes• Recruit HMTs
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HATs, HDACs and HMTsEuchromatin Heterochromatin
HATs HDACs HMTs
Acetyl-lysine trimethyl-lysine+
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DNMT
Txn-level control• Co-repression
– DNA methylation: recruit DNA methyltransferases (DNMTs)– Methylated DNA serves as binding sites for proteins (MeCP2
that recruit HDACs and HMTs
HDAC
HMT
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Processing-level control of gene expression• Alternative splicing• Exonic Splicing Enhancers
– ESE binding proteins• Cell-type specific
Fn
+ ESE Binding proteins
- ESE Binding proteins
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Tln-level control of gene expression• mRNA localization
– Bicoid @ anterior– Oskar @ posterior
• Beta-actin mRNA at leading edge of a migrating fibroblast
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Tln-level control of gene expression• mRNA translation
– Masking by specific proteins that bind to 5’ and 3’ UTR sequences
– Response element is an RNA sequence
– Regulatory Protein binding subject to allosteric control
- Iron = binds and inhibits+ Iron = no binding
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Tln-level control of gene expression• mRNA stability
– polyA tail length 200nt --> 30nt (destroyed)
– Specific sequence effects• 5’-CCUCC-3’ stabilizing (factors bind to mediate this)• 5’-AUUUA-3’ destabilizing (factors bind to mediate this)
– Just one of these can reduce ½-life from 10hrs to 90 minutes
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Tln-level control of gene expression• mRNA stability
– polyA tail length 200nt --> 30nt (destroyed)– Decapping enzyme– 5’ 3’ exonuclease