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1、Yang-Ming Life Science 1 1-2-2004 Control of Eucaryotic Gene ExpressionSteps 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
2、 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 phaseThe control of the cell cycleTo grow or not to grow?That is the question!How to control the cell cycle progression? Accelerator: master s
3、witch 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 checkerExtrinic signalsPhosphorylation site of protei
4、nAlso 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 compo
5、sed of regulatory subunits called “cyclins” and catalytic subunits called “cyclindependent kinases, CDKs” CDKs can associate with different cyclins The cyclin determines which substrates are phosphorylatedIntrinsic master switch to respond extrinsic signal: two key components (cyclin and cdc kinase)
6、.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 bas
7、is of Cdk activationA simplified view of the core of the cell cycle control systemUniqueness 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 gen
8、e 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 regulationAdvantage to use yeast as a mode
9、l system to study gene regulation:1, fungi, closely related to animal2, easy to grow as a bacterium3, can divide in either the haploid or the diploid state4, in the haploid state, mutants are very easy to isolateIsolation many mutants with same phenotype(Wild type)(Mutants)Phenotype analysis of Gal
10、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 muta
11、nt 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?
12、 Uninduced?Answer: uninducedModel 1 is favoredHow to identify regulatory sequence in the promoter region?Gal4 binding siteNew repressor site?TATA boxModular properties of Transcription activators The Gal4 protein performs two different functions. The protein must be able to recognize and to bind spe
13、cifically 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.How can a DNA binding protein recognize the specific sequence in a double strand DNA molecu
14、le without opening the double strand of DNA? Gal4 recognizes the following sequence in the promoter region of Ga11 gene in yeast:CGGAGGACTGTCCTCCGGCCTCCTGACAGGAGGCCro repressor binds to DNAThe DNA binding helix-turn helix motifHow the gene expression can be regulated!Control of RNA splicingPost tran
15、slation modificaiton:Phosphorylation;Acetylation;ADP ribosylation etc.A model for nonsense mediated mRNA decayEnhancer vs upstream regulated sequence (URS) Enhancer: Orientation independent Up or downstream independent Act at very long distance (10-100 Kb) Upstream regulated sequence: Orientation de
16、pendent Only acts at Upstream of promoter Act at short to medium distance Transcriptional synergyHow the transcriptional factor can be regulated?How does the repressor work!Integration at a promoterLocal alteration in chromatin structure directed by “activators”How DNA methylation patterns are faithfully inheritedHow DNA methylation ma
