structure of the nucleus nucleoli: rdna, rrna synthesis, ribosome assembly chromatin: genomic dna -...

Structure of the nucleus• Nucleoli: rDNA, rRNA synthesis, ribosome assembly• Chromatin: Genomic DNA - protein complexes, transcription• Nuclear envelope

– Two membranes (10-50nm separation)

Structure of the nucleus• Nucleoli: rDNA, rRNA synthesis, ribosome assembly• Chromatin: Genomic DNA - protein complexes, transcription• Nuclear envelope

– Two membranes (10-50nm separation)

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

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

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

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

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

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

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)

Chromosomes and chromatin• Chromatin = DNA + associated proteins

– Histone octamer• ( H2A, H2B, H3, H4 ) x2

– Nucleosome = histone octamer + 146 bp DNA

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

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

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

+

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

Euchromatin & heterochromatin• X-inactivation

– Convert one X chromosome to facultative heterochromatin– Random event early in development– Stably maintained through subsequent cell divisions

Before inactivation

AfterXp

XmXp

Xm

Xp

XmXp

XmXp

XmXp

Xm

Xp

XmXp

Xm

Xp

Xm

Xp

XmXp

Xm

Xp

XmXp

XmXp

XmXp

Xm

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

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

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

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

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

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

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

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

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!

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

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

Gene expression: eukaryotes

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

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

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

ENHANCEINSULATE

PEPCK gene

Gene expression: eukaryotes• TXN-level control

– Mechanism• Co-activation

– Co-operative binding between Activator and GTFs– Histone modification: recruit HAT enzymes

Gene expression: eukaryotes• TXN-level control

– Mechanism• Co-repression

– Antagonistic binding: block GTFs

– Histone modification» recruit Histone

deacetylase (HDAC) enzymes

» Recruit HMTs

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

Gene expression: eukaryotes• Processing-level control

– Alternative splicing– Exonic Splicing Enhancers

• ESE binding proteins– Cell-type specific– Fibroblast vs

Hepatocyte

– (RNA editing too)

Fn

Gene expression: eukaryotes• TLN-level control

– mRNA localization• Bicoid @ anterior• Oskar @ posterior

• Beta-actin mRNA at leading edge of a migrating fibroblast

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

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

Gene expression: eukaryotes• TLN-level control

– mRNA stability• polyA tail length 200nt --> 30nt (destroyed)• Decapping enzyme• 5’ 3’ exonuclease