Transcript ch12 FA 11 - Cal State LA

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
(bind beta)
GTP
Ran-GDP
GNEF
GAP
Ran-GDP
(release
beta)
GDP
Pi
Ran-GTP
Ran-GTP
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
Lysine
Acetyl-lysine
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
+
Lysine
trimethyl-lysine
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
Xp
Xm
subsequent cell divisions
Xp
Xm
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
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
INSULATE
PEPCK gene
ENHANCE
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
Fn
– (RNA editing too)
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