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 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

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 Allosteric effector inactivates Lac Repressor DNA binding

6. 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

7. 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

8. 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

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

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

12. 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

13. 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

14. Hutchinson-Gilford Progeria Syndrome Caused by mutations in Lamin A –Premature aging –Most die by age 13 Molecular phenotype is abnormal nuclei shape

15. 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 view with gold particles

16. Molecular weights (MW) Most ions are very small –Na + 23g/mol –Cl - 35g/mol –K + 39g/mol –Mg 2+ 24g/mol –Ca 2+ 40g/mol –PO4 3- 95g/mol Amino acids (AAs) sizes –Glycine75g/mol –Tryptophan204g/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

17. 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

18. 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

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

20. Gene expression: eukaryotes

21. Chromosomes and chromatin Chromatin = DNA + associated proteins –Histone octamer ( H2A, H2B, H3, H4 ) x2 –Nucleosome = histone octamer + 146 bp DNA

22. 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

24. 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-lysine Lysine

25. 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-lysine Lysine +

26. 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 (X b ) versus Orange allele (X o ) Female calico cats have one X b allele and one X o allele Random inactivation of X b or X o yields orange or black patches Cloning of a calico cat confirmed the random nature of X-inactivation

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

28. 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

29. 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

30. 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 ENHANCE INSULATE PEPCK gene

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

32. Txn-level control Co-activation –Nucleosome remodeling: recruit chromatin remodeling complexes

33. Txn-level control Co-activation –Nucleosome remodeling: recruit chromatin remodeling complexes

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

36. Txn-level control Co-repression –Antagonistic binding: block GTFs –Histone modification recruit Histone deacetylase (HDAC) enzymes

37. Txn-level control Co-repression –Antagonistic binding: block GTFs –Histone modification recruit Histone deacetylase (HDAC) enzymes Recruit HMTs

38. HATs, HDACs and HMTs EuchromatinHeterochromatin HATs HDACs HMTs Acetyl-lysinetrimethyl-lysine +

39. 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

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

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

42. 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

43. 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

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