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Regulation of Gene Expression

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 )

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Regulation of Gene Expression

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  1. 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

  2. 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 aTranscriptional (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

  3. 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

  4. 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 Allostericeffector inactivates Lac Repressor DNA binding

  5. 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

  6. 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 Allostericeffector needed for effective DNA binding

  7. 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 Allostericeffector needed for effective DNA binding

  8. Structure of the nucleus • Nucleoli: rDNA, rRNA synthesis, ribosome assembly • Chromatin: Genomic DNA - protein complexes, transcription • Nuclear envelope • Two membranes (10-50nm separation)

  9. Structure of the nucleus • Nucleoli: rDNA, rRNA synthesis, ribosome assembly • Chromatin: Genomic DNA - protein complexes, transcription • Nuclear envelope • Two membranes (10-50nm separation)

  10. 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

  11. 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

  12. Hutchinson-Gilford Progeria Syndrome • Caused by mutations in LaminA • Premature aging • Most die by age 13 • Molecular phenotype is abnormal nuclei shape

  13. The Nuclear Pore Complex (NPC) view from cytoplasm • 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 nucleus side view with gold particles

  14. 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

  15. 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

  16. 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

  17. 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 (bind beta) GTP GDP Ran-GDP Ran-GTP GNEF GAP Ran-GDP Ran-GTP (release beta) Pi

  18. Gene expression: eukaryotes

  19. Chromosomes and chromatin • Chromatin = DNA + associated proteins • Histoneoctamer • ( H2A, H2B, H3, H4 ) x2 • Nucleosome = histoneoctamer + 146 bp DNA

  20. 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

  21. Euchromatin & heterochromatin • Euchromatin • Dispersed, not compacted • Readily accessed by TXN factors and RNAp • Transcriptionally active • Histone modifications • HistoneAcetyltransferase 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 Lysine Acetyl-lysine

  22. Euchromatin & heterochromatin • Heterochromatin • Highly compacted • Not readily accessed by TXN factors or RNAp • Transcriptionally inactive • Histone modifications • HistoneMethylransferase 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 + Lysine trimethyl-lysine

  23. 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 haveone 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

  24. X Chromosome inactivation Xb Xo • Convert one X chromosome to facultative heterochromatin • Random event early in development • Stably maintained through subsequent cell divisions Before inactivation After female male Xb Xo Xb Xo Xb Xo Xb Xo Xb Xo Xb Xo Xb Xo Xb Xo Xb Xo Xb Xo Xb Xo Xb Xo Xb Xo Xb Xo

  25. 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

  26. 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

  27. 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 INSULATE ENHANCE PEPCK gene

  28. Txn-level control • Co-activation • Co-operative binding between Activator and GTFs • Histone modification: recruit HAT enzymes

  29. Txn-level control • Co-activation • Nucleosome remodeling: recruit chromatin remodeling complexes

  30. Txn-level control • Co-activation • Nucleosome remodeling: recruit chromatin remodeling complexes

  31. Txn-level control • Co-activation • Cleared promoter region now accessible to TFIID, other GTFs and RNApII

  32. Txn-level control • Co-repression • Antagonistic binding: block GTFs • Histone modification • recruit Histonedeacetylase (HDAC) enzymes

  33. Txn-level control • Co-repression • Antagonistic binding: block GTFs • Histone modification • recruit Histonedeacetylase (HDAC) enzymes • Recruit HMTs

  34. HATs, HDACs and HMTs Euchromatin Heterochromatin HATs HDACs HMTs + Acetyl-lysine trimethyl-lysine

  35. Txn-level control • Co-repression • DNA methylation: recruit DNA methyltransferases(DNMTs) • Methylated DNA serves as binding sites for proteins (MeCP2 that recruit HDACs andHMTs HDAC HMT DNMT

  36. Processing-level control of gene expression • Alternative splicing • Exonic Splicing Enhancers • ESE binding proteins • Cell-type specific Fn + ESE Binding proteins - ESE Binding proteins

  37. Tln-level control of gene expression • mRNA localization • Bicoid @ anterior • Oskar @ posterior • Beta-actin mRNA at leading edge of a migrating fibroblast

  38. 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

  39. 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

  40. Tln-level control of gene expression • mRNA stability • polyA tail length 200nt --> 30nt (destroyed) • Decapping enzyme • 5’  3’ exonuclease

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