Lecture 15 - Gene Exp in Eukaryotes I PDF

BIOL 366 Lecture 15 Gene Expression in Eukaryotes I - The yeast Gal operon and its applications - Regulatory mechanism unique to eukaryotes Text Section: 21.1.1 – 21.3 Slides will be updated before lecture on Tuesday Announcements: 1. Exam III on Nov 22 with a review / questions...

BIOL 366 Lecture 15 Gene Expression in Eukaryotes I - The yeast Gal operon and its applications - Regulatory mechanism unique to eukaryotes Text Section: 21.1.1 – 21.3 Slides will be updated before lecture on Tuesday Announcements: 1. Exam III on Nov 22 with a review / questions on Nov 20 Is cumulative: Covers lectures 1 – 15 Format: like midterm II; Includes multiple choice questions, and written answer questions 60 min long similar to midterm II 2. Review next Tuesday 4. 2-3 more assignments on the remaining lectures 5. Final will be cumulative, with approximately: - 50% on material covered after Exam II - 50% on material before Exam II (all lecture equal weight) 2 Eukaryotic promoters vary in complexity More complex organisms have more complex promoters more control elements changes in gene expression during development intercellular communication interaction with the environment, etc. 3 All three eukaryotic RNA Polymerases: have little/no affinity for their promoters. almost always require activator & coactivator proteins. Regulatory Proteins that control transcription in eukaryotes General Transcription Factors DNA-binding Transcription Activators (Transactivators) DNA-binding Transcription Coactivators Repressors: (sometimes used) FIGURE 21-3: 4 Activation of eukaryotic genes involves: General (basal) transcription factors: Proteins that: Are required at every Pol II promoter Bind promoter region (near gene) Example: TATA binding Protein (TBP) Text Fig 21-5a 5 Activation of eukaryotic genes involves: Activators: proteins that: Bind distant regulatory sites (aka) Enhancers in higher eukaryotes Upstream Activating Sequences (UAS) in yeast Recruit Pol II complex to the promoter Example: Gal4 of yeast Coactivators: proteins that: Are required at essentially all promoters Function as a bridge between activators and RNA Pol Do not interact with DNA directly Example: Mediator & TFIID 6 HMG (High Mobility) proteins bring distant DNA regulatory elements near the promoter. They bend DNA, helping form loops between enhancer and promoter elements. Binding is nonspecific. HMG proteins have a helix-loop-helix type DNA binding domain (HMG-box). They act as architectural Transcription Factors. Text Fig 21.6. Binding of the DNA- binding domain of the HMG-D protein Text Fig 21-5a of Drosophila, to its DNA target. 7 Repression of eukaryotic gene expression. Eukaryotes use repressors; proteins that can: Bind specific DNA sequences and inhibit transcription. OR Disrupt / prevent contacts between Pol II and activators or coactivators. Text Fig 21-5b 8  Utility of The yeast GAL expression system. (the Yeast Two-hybrid assay) Background: 1. GAL genes enable yeast to take in and metabolize galactose 2. There are four structural Gal genes (GAL1, GAL10, GAL2 and GAL7) 4. GAL structural genes are on different loci within the yeast genome 5. GAL structural genes are controlled by the same regulatory proteins 3. There are three regulatory Gal genes: ▪ GAL4: a DNA-binding transcription activator (aka transactivator) ▪ GAL80: a repressor ▪ GAL3: ligand (galactose) sensor 9  Expression from the GAL genes The promoters for the GAL genes consist of: - A TATA box - One or more upstream activator sequence (UASGAL) - Each UASGAL site is recognized by the Gal4 protein (Gal4p), a DNA-binding transactivator. 10 Roles of Gal3p, Gal4p, and Gal80p in the regulation of GAL genes. (a) No galactose: Gal4p binds UASGAL Gal80p binds Gal4p and prevents its activation of Pol II. (a) Galactose present: Lactose acts as effector & activates Gal3p Activated Gal3p binds Gal80p, alters its conformation, and inactivates it. Gal4p now can bind Pol II and activate transcription. More on Gal4p: Gal4p activates all structural GAL genes Gal4p has two domains: – a DNA binding – an RNA Pol activating domain Both Gal4p domains are active on their own. However, they MUST BE in close proximity to activate transcription Yeast Two-hybrid assay (aka Y-2-H assay/system) Is based on ability of Gal4 protein (Gal4p) to drive expression of yeast GAL genes Is used to detect protein-protein interactions (e.g., proteins X and Y) To perform a two hybrid analysis 1. A reporter gene is placed under the control of the GAL promoter and introduced in yeast genome Text Fig 7-28 2. The a DNA binding domain of Gal4p is fused to a protein (protein X) 3. The RNA Pol activating domain of Gal4p is fused to a second protein (protein Y) 4. If protein X and protein Y interact, the DNA binding and activation domains of Gal4p are brought together and can activate expression from the GAL promoter 13 Some TFs form functional Heterodimers 14  Heterodimeric TFs Most eukaryotic TFs bind to DNA as homodimers. However, several types of structurally related eukaryotic transcription factors can form heterodimers A hypothetical family of four different but structurally related proteins could form up to 10 different dimeric species Each dimer could have a unique activity 15  Heterodimeric TFs Example: The mammalian AP-1 TFs (e.g., Fos, Jun) AP-1 transcription factors control cell proliferation and differentiation The protein-dimerization and DNA-binding regions of AP-1 family members are of the basic leucine zipper type. 16 Structural model of the AP-1 heterodimer of Fos (purple) and Jun (green), bound to DNA. Binding of TF to its target DNA was detected by Mobility Shift Assay (a) DNA fragment containing the AP-1–binding site was labeled with 32P. (b) The labeled DNA was mixed with Fos, FosC (a fragment of Fos), or Jun, or with a mixture of Fos and Jun or a mixture of FosC and Jun. (a) Reactions were analyzed by polyacrylamide gel electrophoresis, then autoradiography. (b) Binding of protein dimers to the DNA causes the complex to migrate more slowly through the gel, resulting in distinct bands. Results: Fos and FosC can bind DNA target as a heterodimer and not as a monomer, or homodimer Fig. 21-13 Fos and Jun can 17 function as heterodimers Question: Assume that you have access to the cDNA encoding the transcription factor (TF-x), and the Genomic DNA fragment for Gene-Y. How would you use the Mobility Shift Assay to determine if TF-x binds the promoter region for gene Y? - Obtain some TF-X protein (purify from organism or produce the recombinant form) - Label the genomic DNA fragment for Gene Y with 32P or a detectable label - Mix TF-x with the labeled DNA and run on a gel - Visualize the signal to see if DNA mobility was affected. If mobility was affected, there is interaction between TF-x and Gene-Y DNA 18 Two regulatory mechanism unique to eukaryotes 19  Mechanism 1: Genetic Imprinting In most diploid cells, both homologous genes (i.e., both alleles of a gene) are expressed equally. Some higher eukaryotes, however, have mechanisms to turn on/off the expression of an allele derived from one parent, via imprinting. Imprinting is: - Is an inheritance process independent of Mendelian inheritance - Epigenetic in nature; involves methylation of cytosine residues - Affected genes are expressed in a parent-of-origin-specific manner - Is observed in fungi, plants and animals 20  Mechanism of Imprinting: An example (See Fig. 21-18 ) Imprinting of the mammalian IGF2 gene. CTC-binding factor (CTCF) binds the insulator of IGF2. This enables insulator function and turns off transcription from IGF2 Methylation of insulator sequences prevents CTCF binding to DNA. IGF2 gene can now be expressed 21  Mechanism 2: Dosage Compensation of sex chromosome genes 22 Dosage Compensation I Balanced Gene Expression from Sex Chromosomes is achieved via three mechanisms. A) Barr body formation In mammals, one X chromosome of the female (XX) is inactivated by forming a compact structure called a Barr body. 23 Dosage Compensation II B) Stronger expression of certain genes In Drosophila, the single X chromosome of the male (XY) is transcribed at twice the level of the X chromosomes in the female (XX). Fig 21-19 b 24  Dosage Compensation III C) Weaker transcription of certain genes In C. elegans, the two X chromosomes of the hermaphrodite (XX) are transcribed at half the level of the single X chromosome of the male (X0). Fig 21-19 c 25