Cell Bio Good
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Yang-Ming Life Science 1 – 1-2-2004
• Control of Eucaryotic Gene Expression
Steps in the cycle • At the beginning, dividing cells exist in G1, the period when cells make the decision as to whether or not they can divide. • If signals, growth conditions, etc. not favorable cells enter quiescent state, G0 • Next step is to double DNA content, called S phase • Components for chromosomal division (mitosis) and cell division (cytokinesis) made during G2 • Cells divide in M phase
The control of the cell cycle
To grow or not to grow? That is the question!
How to control the cell cycle progression? • Accelerator: master switch gene to relief brake and turn on the engine! push cell to divide! – growth factor and its receptors – G1cell division kinase (cdk) – G2 cell division kinase
• Brake: stop cell division before things are ready! – tumor suppressor genes
• Checkpoint – Gate checker
Extrinic signals
Phosphorylation site of protein
Also occur at threonine, tyrosine or histidine
Introduce a negative charge and a high energy bone. That may change the conformation of the protein, then its biological activity, such as enzyme activity.
Master Switches for Cell Cycle • Heterodimeric kinases regulate the process • These enzymes are composed of regulatory subunits called “cyclins” and catalytic subunits called “cyclindependent kinases, CDKs” • CDKs can associate with different cyclins • The cyclin determines which substrates are phosphorylated
Intrinsic master switch to respond extrinsic signal: two key components (cyclin and cdc kinase).
Cyclins and CDKs • 3 groups of cyclins: levels rise and fall with cell cycle stages – G1 cyclins: D and E – S-phase cyclins: A and E – M-phase cyclins: A and B
• 3 groups fo CDKs: levels remain fairly constant – G1 CDKs: Cdk2, Cdk4, Cdk6 – S-phase CDKs: Cdk2 – M-phase CDKs: Cdk1 (also called Cdc2)
The structure basis of Cdk activation
A simplified view of the core of the cell cycle control system
Uniqueness of eukaryotic gene regulation • genes are not organized into operons — each gene has its own promoter. • Usually eukaryotic regulatory genes are not linked to the genes that they regulate. • Proteins involved in gene regulation must be compartmentalized into the cell nucleus. • Eukaryotic DNA is wrapped around nucleosomes to construct a protein DNA scaffold known as chromatin. Changes in chromatin structure can influence regulation.
Budding yeast as a model to study gene regulation
Advantage to use yeast as a model system to study gene regulation: 1, fungi, closely related to animal 2, easy to grow as a bacterium 3, can divide in either the haploid or the diploid state 4, in the haploid state, mutants are very easy to isolate
Isolation many mutants with same phenotype
(Wild type) (Mutants)
Phenotype analysis of Gal mutant • The first mutant isolated was Gal4 – which gives uninducible expression of the Gal genes. • Heterozygous diplioids constructed by mating a Gal4 – to wild type (Gal4 –/ Gal4+) have normal regulation. • Gal4 – is recessive. • Gal4 is a positive regulator of Gal gene expression.
Phenotype analysis of Gal mutant • Gal80 –, which gives constitutive expression of the Gal genes. • Gal80 –/Gal80+ heterozygous diploids show normal regulation. • Gal80 – is recessive. • The normal function of Gal80 is to negatively regulate the Gal genes.
What happen if both Gal4 and Gal80 gene are mutated? Regulated? Constitutive expression? Uninduced?
Answer: uninduced Model 1 is favored
How to identify regulatory sequence in the promoter region?
Gal4 binding site
New repressor site?
TATA box
Modular properties of Transcription activators • The Gal4 protein performs two different functions. • The protein must be able to recognize and to bind specifically to the DNA of the UAS (Upstream Activation Sequence) sequence in the Gal gene promoters. • The protein must contain a region that can interact with RNA polymerase to stimulate transcription initiation.
Gal4 recognizes the following sequence in the promoter region of Ga11 gene in yeast: CGGAGGACTGTCCTCCG GCCTCCTGACAGGAGGC How can a DNA binding protein recognize the specific sequence in a double strand DNA molecule without opening the double strand of DNA?
Cro repressor binds to DNA
The DNA binding helix-turn helix motif
How the gene expression can be regulated!
Control of RNA splicing Post translation modificaiton: Phosphorylation; Acetylation; ADP ribosylation etc.
A model for nonsense mediated mRNA decay
Enhancer vs upstream regulated sequence (URS) • Enhancer: – Orientation independent – Up or downstream independent – Act at very long distance (>10-100 Kb)
• Upstream regulated sequence: – Orientation dependent – Only acts at Upstream of promoter – Act at short to medium distance
Transcriptional synergy
How the transcriptional factor can be regulated?
How does the repressor work!
Integration at a promoter
Local alteration in chromatin structure directed by “activators”
How DNA methylation patterns are faithfully inherited
How DNA methylation may help turn off genes
How is the mRNA degraded?
Translation control
• Control of Eucaryotic Gene Expression
Steps in the cycle • At the beginning, dividing cells exist in G1, the period when cells make the decision as to whether or not they can divide. • If signals, growth conditions, etc. not favorable cells enter quiescent state, G0 • Next step is to double DNA content, called S phase • Components for chromosomal division (mitosis) and cell division (cytokinesis) made during G2 • Cells divide in M phase
The control of the cell cycle
To grow or not to grow? That is the question!
How to control the cell cycle progression? • Accelerator: master switch gene to relief brake and turn on the engine! push cell to divide! – growth factor and its receptors – G1cell division kinase (cdk) – G2 cell division kinase
• Brake: stop cell division before things are ready! – tumor suppressor genes
• Checkpoint – Gate checker
Extrinic signals
Phosphorylation site of protein
Also occur at threonine, tyrosine or histidine
Introduce a negative charge and a high energy bone. That may change the conformation of the protein, then its biological activity, such as enzyme activity.
Master Switches for Cell Cycle • Heterodimeric kinases regulate the process • These enzymes are composed of regulatory subunits called “cyclins” and catalytic subunits called “cyclindependent kinases, CDKs” • CDKs can associate with different cyclins • The cyclin determines which substrates are phosphorylated
Intrinsic master switch to respond extrinsic signal: two key components (cyclin and cdc kinase).
Cyclins and CDKs • 3 groups of cyclins: levels rise and fall with cell cycle stages – G1 cyclins: D and E – S-phase cyclins: A and E – M-phase cyclins: A and B
• 3 groups fo CDKs: levels remain fairly constant – G1 CDKs: Cdk2, Cdk4, Cdk6 – S-phase CDKs: Cdk2 – M-phase CDKs: Cdk1 (also called Cdc2)
The structure basis of Cdk activation
A simplified view of the core of the cell cycle control system
Uniqueness of eukaryotic gene regulation • genes are not organized into operons — each gene has its own promoter. • Usually eukaryotic regulatory genes are not linked to the genes that they regulate. • Proteins involved in gene regulation must be compartmentalized into the cell nucleus. • Eukaryotic DNA is wrapped around nucleosomes to construct a protein DNA scaffold known as chromatin. Changes in chromatin structure can influence regulation.
Budding yeast as a model to study gene regulation
Advantage to use yeast as a model system to study gene regulation: 1, fungi, closely related to animal 2, easy to grow as a bacterium 3, can divide in either the haploid or the diploid state 4, in the haploid state, mutants are very easy to isolate
Isolation many mutants with same phenotype
(Wild type) (Mutants)
Phenotype analysis of Gal mutant • The first mutant isolated was Gal4 – which gives uninducible expression of the Gal genes. • Heterozygous diplioids constructed by mating a Gal4 – to wild type (Gal4 –/ Gal4+) have normal regulation. • Gal4 – is recessive. • Gal4 is a positive regulator of Gal gene expression.
Phenotype analysis of Gal mutant • Gal80 –, which gives constitutive expression of the Gal genes. • Gal80 –/Gal80+ heterozygous diploids show normal regulation. • Gal80 – is recessive. • The normal function of Gal80 is to negatively regulate the Gal genes.
What happen if both Gal4 and Gal80 gene are mutated? Regulated? Constitutive expression? Uninduced?
Answer: uninduced Model 1 is favored
How to identify regulatory sequence in the promoter region?
Gal4 binding site
New repressor site?
TATA box
Modular properties of Transcription activators • The Gal4 protein performs two different functions. • The protein must be able to recognize and to bind specifically to the DNA of the UAS (Upstream Activation Sequence) sequence in the Gal gene promoters. • The protein must contain a region that can interact with RNA polymerase to stimulate transcription initiation.
Gal4 recognizes the following sequence in the promoter region of Ga11 gene in yeast: CGGAGGACTGTCCTCCG GCCTCCTGACAGGAGGC How can a DNA binding protein recognize the specific sequence in a double strand DNA molecule without opening the double strand of DNA?
Cro repressor binds to DNA
The DNA binding helix-turn helix motif
How the gene expression can be regulated!
Control of RNA splicing Post translation modificaiton: Phosphorylation; Acetylation; ADP ribosylation etc.
A model for nonsense mediated mRNA decay
Enhancer vs upstream regulated sequence (URS) • Enhancer: – Orientation independent – Up or downstream independent – Act at very long distance (>10-100 Kb)
• Upstream regulated sequence: – Orientation dependent – Only acts at Upstream of promoter – Act at short to medium distance
Transcriptional synergy
How the transcriptional factor can be regulated?
How does the repressor work!
Integration at a promoter
Local alteration in chromatin structure directed by “activators”
How DNA methylation patterns are faithfully inherited
How DNA methylation may help turn off genes
How is the mRNA degraded?
Translation control
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