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

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

3. Structure of the nucleus
Nucleoli: rDNA, rRNA synthesis, ribosome assembly
Chromatin: Genomic DNA - protein complexes, transcription
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
Mutations in Lamin A
linked to disease
Molecular phenotype
is misshapen nuclei

4. Structure of the nucleus
Nucleoli: rDNA, rRNA synthesis, ribosome assembly
Chromatin: Genomic DNA - protein complexes, transcription
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
Mutations in Lamin A
linked to disease
Molecular phenotype
is misshapen nuclei

5. Structure of the nucleus
Nucleoli: rDNA, rRNA synthesis, ribosome assembly
Chromatin: Genomic DNA - protein complexes, transcription
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
Mutations in Lamin A
linked to disease (HGPS)
Molecular phenotype
is misshapen nuclei

6. Structure of the nucleus
Nuclear envelope
Gated by nuclear pore complex (NPC)
15-30 times larger than a ribosome
Composed of ~30 nucleoporin proteins
Exhibits 8-fold symmetry
Small molecules (< 40 kD) diffuse freely

7. 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

8. 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

9. 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

10. Regulation of nuclear import/export
mRNA export tied to splicing
EJC bridges to NPC
Bridging proteins (Aly, TAP) release after export
EJC release after TLN

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

12. 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

14. Euchromatin & heterochromatin
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
Lysine
Acetyl-lysine

15. Euchromatin & 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 +
Lysine
trimethyl-lysine

16. Euchromatin & heterochromatin
X-inactivation
Males have only 1 X Chromosome
Females have 2 X Chromosomes
Gene dosage in females is regulated by only using 1 of the 2 available X chromosomes
Cats have a pigment gene on X
Black allele vs orange allele
Female calico cats have random patches of black vs orange fur
Cloning of a calico cat confirmed the random nature of X-inactivation

17. Euchromatin & heterochromatin
X-inactivation
Convert one X chromosome to facultative heterochromatin
Random event early in development
Stably maintained through
subsequent cell divisions
Before inactivation
After Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm

18. Heterochromatin & euchromatin
X-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

19. Heterochromatin & euchromatin
Examples of factors that specifically bind various chromatin modifications

20. Heterochromatin & euchromatin
The Nucleus as an Organized Organelle
Chromosome ordering is directed by the nuclear envelope proteins.
In the nucleus, mRNAs are synthesized at discrete sites.
Estrogen Receptor txn of:
GREB1 gene on Chr 2
TRFF1 gene on Chr 21
- Estrogen (E2) = separate
+ Estrogen (E2) = together

21. Heterochromatin & euchromatin
The Nucleus as an Organized Organelle
Transcription occurs in distinct locations, “transcription factories”
Genes from different chromosomal locations are brought together
DNA sequences that participate in a common biological response but reside on different chromosomes interact within the nucleus.
Nuclear Matrix
May function as scaffold
for organization

22. 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 txn
mRNA and protein levels slowly return to original level

23. 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 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

24. Gene expression: bacteria
Inducible gene expression
Example: Sugar catabolism
In the absence of lactose, no need to have enzymes that metabolize it
- Lactose, Lac Repressor binds Operator and blocks TXN

25. 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

27. 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
Look for -35 (TTGACA) and -10 (TATAAT) elements!

28. 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
Tryptophan, Trp Repressor can’t bind Operator
Allosteric effector needed for effective DNA binding

29. 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

31. Gene expression: eukaryotes

32. 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

33. Gene expression: eukaryotes
How do we identify promoter “elements” important for gene expression?
Deletion Mapping
DNA footprinting
Using DNase I digestion
Genome-wide location analysis
Using chromatin immunoprecipitation

34. Gene expression: eukaryotes
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

35. Gene expression: eukaryotes
TXN-level control
Mechanism
Co-activation
Co-operative binding between Activator and GTFs
Histone modification: recruit HAT enzymes

36. Gene expression: eukaryotes
TXN-level control
Mechanism
Co-activation
Nucleosome remodeling: recruit chromatin remodeling complexes

39. Gene expression: eukaryotes
TXN-level control
Mechanism
Co-repression
Antagonistic binding: block GTFs
Histone modification
recruit Histone deacetylase (HDAC) enzymes
Recruit HMTs

40. Gene expression: eukaryotes
TXN-level control
Mechanism
Co-repression
DNA methylation: recruit DNA methyltransferases (DNMTs)
Methylated DNA serves as binding sites for proteins (MeCP2)
Recruit… HDACs, HMTs

41. Gene expression: eukaryotes
Processing-level control
Alternative splicing
Exonic Splicing Enhancers
ESE binding proteins
Cell-type specific
Fibroblast vs Hepatocyte
(RNA editing too) Fn

42. Gene expression: eukaryotes
TLN-level control
mRNA localization
anterior
posterior
Beta-actin mRNA at leading edge of a migrating fibroblast

43. Gene expression: eukaryotes
TLN-level control
mRNA translation
Masking by specific proteins that bind to 5’-/3’-UTR sequences
IRE is an RNA sequence
IRP binds to IRE
- Iron = bind and inhibit
+ Iron = no bind

44. Gene expression: eukaryotes
TLN-level control
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

45. Gene expression: eukaryotes
TLN-level control
mRNA stability
polyA tail length 200nt --> 30nt (destroyed)
Decapping enzyme
5’  3’ exonuclease