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

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

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

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

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

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

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• 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

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

Hutchinson-Gilford Progeria Syndrome• Caused by mutations in

Lamin A– Premature aging– Most die by age 13

• Molecular phenotype is abnormal nuclei shape

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 viewwith goldparticles

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

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-GDPGTP GDP

Ran-GTPRan-GDP

Pi

GNEF

GAP

(bind beta)

(release beta)

Gene expression: eukaryotes

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+

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 have one 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

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

Before inactivation

After

Xb

XoXb

Xo

Xb

XoXb

XoXb

XoXb

Xo

Xb

XoXb

Xo

Xb

Xo

Xb

XoXb

Xo

Xb

XoXb

XoXb

XoXb

Xo

female male

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

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

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

Txn-level control• Co-activation

– Co-operative binding between Activator and GTFs

– Histone modification: recruit HAT enzymes

Txn-level control• Co-activation

– Nucleosome remodeling: recruit chromatin remodeling complexes

Txn-level control• Co-activation

– Nucleosome remodeling: recruit chromatin remodeling complexes

Txn-level control• Co-activation

– Cleared promoter region now accessible to TFIID, other GTFs and RNApII

Txn-level control• Co-repression

– Antagonistic binding: block GTFs

– Histone modification• recruit Histone deacetylase

(HDAC) enzymes

Txn-level control• Co-repression

– Antagonistic binding: block GTFs

– Histone modification• recruit Histone deacetylase

(HDAC) enzymes• Recruit HMTs

HATs, HDACs and HMTsEuchromatin Heterochromatin

HATs HDACs HMTs

Acetyl-lysine trimethyl-lysine+

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

Processing-level control of gene expression• Alternative splicing• Exonic Splicing Enhancers

– ESE binding proteins• Cell-type specific

Fn

+ ESE Binding proteins

- ESE Binding proteins

Tln-level control of gene expression• mRNA localization

– Bicoid @ anterior– Oskar @ posterior

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

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

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

Tln-level control of gene expression• mRNA stability

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