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MCB Lecture 3 – Gene Regulation What is an inducible gene? o An inducible gene is normally off, but can be turned on. What is a repressible gene? o A repressible gene is always on, but can be turned off. What are constitutive genes? o Constitutive genes are those that stay more or less constant because they are the housekeeping genes of the cell. What are three ways to measure gene expression? o Northern Blot o RT-PCR o DNA Microarray What is the main determinant for expression of housekeeping genes? o The deviations from the consensus sequence reduces promoter function, leading to differences in mRNA product What are specificity factors? o They alter the specificity of RNA Polymerase for promoter in transcription What is the specificity factor in Bacteria and Eukaryotes? o Bacteria: Sigma Factor o Eukaryotes: TBP (Tata Binding Protein) What is a repressor? What type of regulation is it? o A Repressor impedes access of RNA Polymerase to the promoter. It is negative regulation. What is an activator? What type of regulation is it? o An Activator enhances RNA Polymerase – Promoter interaction. It is positive regulation. What two signal molecules act on a repressor? What do they do? o Co-repressors: bind to Repressor to enhance the binding to the operator so that RNA Polymerase cannot bind to the promoter. o Inducers: Inducers bind to the repressor to prevent it from binding to the operator, so that RNA Polymerase can bind. What two signal molecules act on an activator? What do they do? o Co-Activators: Binds to the activator to enhance the binding of it so that it can help RNA Polymerase bind to the strand. o Repressors: Binds to the activator so that the activator cannot bind to the strand, preventing RNA Polymerase from binding. Do you get transcription when you have: o Co-Activator + Activator? YES o Inducer + Repressor? YES o Co-Repressor + Repressor? NO o Repressor + Activator? NO What is an operon? Where are they found? o Operons are in Prokaryotes only. They are clusters of genes involved in the same biochemical pathway. What is the name of the sequence in the operon where the repressor binds? o Operator What is the product of an operon? o A Polycistronic mRNA LAC OPERON: What type of organism utilizes the Lac Operon? o E. Coli What type of energy source does it prefer? o Glucose Why does it use Lactose as a backup energy source? o Because Lactose is made from Galactose and Glucose. Lactose can be broken down to get Glucose (which they prefer) What does the enzyme Galactoside Permease do? o Allows Lactose into the cell. What does B-Galactosidase do? o It breaks down Lactose into Allolactoe and then Galactose/Glucose Where are the lacZ, lacY, and lacI genes located? o lacZ and lacY are in the operon. lacI is a repressor gene located somewhere else in the genome What is the role of Allolactose? o Allolactose acts as an inducer. It binds to the repressor, so that the repressor is removed from the operon and transcription of the gene can occur by RNA Polymerase cAMP Responsive Protein (CRP or CAP) does what for the Lac Operon? o CRP (CAP) is an activator. cAMP levels are lowered by the presence of Glucose. However, when little Glucose is present, the cAMP levels go up. cAMP acts as a co-activator for CRP (CAP), which then binds to the Lac Operon to activate it. Under these conditions, will the Lac Operon be transcribed? What is present on the strand for each one (activators, repressors, etc.)? o + Glucose, +Lactose = NO Present on Strand: Nothing o +Glucose, -Lactose = NO Present on Strand: No CRP (because high glucose lowers cAMP levels). Because there is no lactose to bind to the repressor (as an inducer), the repressor IS on the strand. Repressor: YES; Activator: NO o –Glucose, -Lactose = NO Present on Strand: There is CRP on the strand because low glucose levels means high levels of cAMP. The cAMP (as a co- activator) binds to CRP, which allows it to bind to the strand. However, the lack of Lactose means that no Allolactose can bind to the repressor (as an inducer), so the repressor is also on the strand. Repressor: YES; Activator: YES o –Glucose, +Lactose = YES Present on Strand: There is CRP on the strand because the low glucose levels means high levels of cAMP. The cAMP (as a coactivator) binds to the CRP, which allows it to bind to the strand. The presence of Lactose allows Allolactose to bind to the repressor (as an inducer). This removes the repressor from the strand and allows transcription to occur. Tryptophan Operon: What is the Trp Operon? o It consists of a promoter region and 5 genes involved in biosynthesis of tryptophan. Which region does the repressor protein recognize? o The Promoter Region - Operator What is the function of Trp? o It acts as a co-repressor to the repressor. Two Trp molecules must be bound to effectively bind to the operator Where is the repressor transcribed? o Elsewhere in the E. Coli genome. It is constitutively expressed (always present) What happens when there is high Trp? o Trp acts as a co-repressor and binds to the repressor, which can then bind to the operator and prevent transcription By how much can the Trp repressor reduce expression? o Factor of 70 By how much can Attenuation decrease expression of Trp operon? o Factor of 10 How much, collectively, can attenuation and Trp Repressor reduce expression of the Trp Operon? o 700 Fold Collectively What is attenuation? o A mode of transcriptional regulation of operons in prokaryotes by prematurely terminating transcription. Where does attenuation happen? o In the Open Reading Frame, which is upstream of the structural genes (14 amino acids) What is a leader peptide? o The sequence of mRNA in the ORF that codes for two Trp’s. What happens in attenuation when there are low levels of Trp? o When there are low levels of Trp, the Leader Peptide stalls at the code for the two Trp’s. This allows the 2:3 hairpin binding, and attenuation is prevented (so the strand can be transcribed and Trp can be made) What happens in attenuation when there are high levels of Trp? o When there are high levels of Trp, the Leader Peptide is able to synthesize the protein quickly because Trp’s are available. Therefore, it moves through quickly and does not give the 2:3 time to hairpin. Because of this, the 3:4 hairpin forms instead, which prevents transcription any further. What type of transcriptional regulation (that was previously discussed) is this? o Rho-Independent Termination (hairpins) Regulation of Eukaryotic Gene Transcription How does the structure of Heterochromatin regulate transcription? o It is too condensed for transcription to happen, so you get no gene expression. How does the structure of Euchromatin regulate transcription? o Euchromatin is sections of the DNA that are unwound and allow transcription to occur. (Only particular regions) What are Hypersensitive Sites? o Regions in chromosomes that are devoid of nucleosomes and allow for transcriptions factors to bind (so genes can be expressed) o They are upstream of the transcription site What is used as a lab tool to degrade DNA (for Hypersensitive Sites). o DNase What are Histone Acetyl Transferases (HATs)? o They covalently modify the core histones of nucleosomes (specifically H3 and H4) by adding acetyl groups and making histones less positive. When the histones are less positive, they have a weaker bond to the negatively charged DNA. o They transfer this to Lysine and Arginine!! What are Histone Deacetylases? o They covalently modify the core histones of nucleosomes (specifically H3 and H4) by removing acetyl groups and making the histones more positive. When the histones are more positive, they have a stronger bond to the negatively charged DNA. What are SWI/SNF Complexes? o They are remodeling proteins that use ATP to remodel nucleosomes by repositioning them or removing them What is Rubinstein-Taybi Syndrome? o Caused by a deficiency of Histone Acetyl Transferase, due to mutation in CBP When Histone Acetyl Transferase is not working properly, it is unable to weaken the bond between DNA and Histone Proteins. This means that those regions cannot be expressed. o Patient presents with: Growth deficiency, microcephaly, broad thumbs, big toes, and mental retardation. What is an enhancer? o It is an element on the DNA where transcription factors can bind to increase the activity of transcription o Can be close (acting in CIS) or come from far away (acting in TRANS) What is an insulator? o They limit the RANGE of enhancers by binding to the DNA and forming looped domains, which act as a physical block. o They decrease the activity of transcription What is a Barrier Sequence? o It prevents spreading of the heterochromatic state of DNA What is Deletion Mapping? o It is used to find regulatory sequences (positive and negative) by deleting particular regions of DNA and looking at the effect on Promoter Activity In Deletion Mapping, what type of deletion causes an increase in Promoter Activity? o A deletion of a repressor In Deletion Mapping, what type of deletion causes a decrease in Promoter Activity? o A deletion of a activator What is needed for RNA Pol II to bind to a promoter? o General Transcription Factors: TFIID, TFIIB, TFIIF, TFIIE, TFIIH o Trans-activators (which bind to enhancers) o Co-activators o Mediator Co-activators What two types of activity does TFIID have? o General Transcription Factor Activity o Co-Activator Activity What is the mechanism by which a Steroid Hormone binds to a Nuclear Hormone Receptor for Gene Activation? o 1. Hormone enters the cell by diffusion o 2. Binds to the hormone receptor in the cytoplasm o 3. Receptor translocates to the nucleus (usually a dimer) o 4. The receptor acts as a transcription factor by binding to the DNA Binding Domain (DBD) in the Major Groove o What is the name of the promoter element that the Receptor binds to? Hormone Response Element (HRE) – Palindromic Repeat o What is an example of a Steroid Hormone that does this? Glucocorticoid What is the mechanism for transcriptional regulation done by Glucocorticoids? o 1. The Glucocorticoid Receptor (R) is normally bound by the HSP 90 and is inactive. o 2. Binding of Glucocorticoid to the Glucocorticoid Receptor releases it from HSP 90, which induces the dimerization of R (RR) o RR moves to the nucleus (as a Dimeric Receptor DBD) o RR activates HRE element through chromatin remodeling coactivators (HATs) o ** When the glucocorticoid is available to bind to the receptor, different transcription factors can lead to different effects of gene expression based on where the receptor binds. How does Tamoxifen treat breast cancer? o It is a competitive antagonist of the estrogen receptor. o It binds to the estrogen receptor as a CO-REPRESSOR o This blocks cell division Protein Phosphorylation and Gene Activation How does Epinephrine act in Gene Activation? o 1. Epinephrine activates G-Protein Coupled Receptors o 2. Activated G-Protein binds to and activates Adenylyl Cyclase o 3. cAMP level increases o 4. cAMP binds to Regulatory Subunit of Protein Kinase A (PKA) o Released PKA Catalytic Subunits move to the nucleus o 5. PKA phosphorylates the cAMP-Response Element Binding Protein (CREB) in the nucleus o 6. CREB binds to cAMP Response Element (CRE) in the Promoters of Target Genes o 7. CREB Binding Protein (CBP) binds as a co-activator o 8. Histone Acetylation and Chromatin Remodeling facilitate Transcription. Co-Repressors: o What type of activity do co-repressors have? Histone Deacetylase (HDAC) Activity This removes acetyl groups so that the Histones tightly bind to the DNA and it cannot be transcribed o Other ways co-repressors work: 1. Histone Methyl Transferases 2. H3-K9 Methylation Lysine Residue #9 of Histone H3 Methylation Decreasing Transcription: Methylation and De-acetylation Where in Eukaryotic DNA can methyl groups be added? o It is limited to Cytosine bases in CpG dinucleotides What is the enzyme called that adds methyl groups? o DNA Methylase Does methylating DNA silent a gene or activate a gene? o Silences it Discuss the MeCP example for Histone Methylation. o 1. MeCP Proteins bind to Methylated CpG nucleotides o 2. MeCP 2 recruits HDAC Activitiy (removed acetyl groups) This induces chromatin remodeling o 3. MeCP2 aids in methylation of H3K9 (Lys9 on Histone H3) o 4. HP1 (Heterochromatin Protein 1) binds to Methylated H3K9 and to itself, condensing the chromatin DNA/Protein Interactions DNA-Protein interactions can depend on two types of bonds – what are they? o Hydrogen Bonding o Hydrophobic Interactions What are the amino acids involved in DNA Binding? o N – Asparagine o Q – Glutamine o E – Glutamate o R – Arginine o K – Lysine What is the DNA target site usually? o A Palindrome What is a Helix-turn-Helix Motif? o A DNA/Protein Interaction that can be found in Bacteria and Humans o It is a helical region interacting with the DNA o Rich in NQERK N – Asparagine Q – Glutamine E – Glutamate R – Arginine K – Lysine What is an example of the Helix-Turn-Helix in bacteria? o The Lac Repressor How does the Helix-turn-Helix function in Humans? o It is found in the homeodomain on transcription factors o It binds to the homeobox on DNA What is an example of Helix-turn-Helix in Humans? o HOXD13 What causes Synpolydactyly? o A mutation on the HOXD13 gene. o The homeobox is mutated (DNA) so transcription factors can not bind What is a homeodomain? o A region on protein that binds to complementary DNA What is a homeobox? o A region on the DNA in which the protein binds What are Zinc Fingers? o A DNA/Protein Interaction in Humans o Zinc Fingers do not directly interact with DNA. They interact with Transcription Factors o Consists of Zinc 2+ions held by 4 Cysteines OR 2 Cysteines and 2 Histines o One Zinc Fingers yields a weak interaction with DNA, but multiple Zinc Fingers increase the strength What is the name of a transcription factor that uses zinc fingers? o TFIIA What is an HMG-Box Motif? o High Mobility Group-Box Motif o Consists of three alpha-helical (protein) regions that interact with DNA o The DNA bends, allowing for interaction with other transcription factors on nearby sites. What is an example of an HMG-Box Motif? o SRY Gene – Responsible for male sexual differentiation and contains HMG Box. Protein-Protein Interactions What are Leucine Zippers? o Leucine-Rich Regions on Transcription Factors that allow for the binding of one transcription factor to another, which increases transcription o Have bZIP domains that interact with DNA What is a Helix-Loop-Helix? o Involved in protein-dimer formation What is an example of a Helix-loop-Helix? o MyoD transcription factors use Helix-Loop-Helix Regulating Translation How does negative feedback affect translation in ribosomal protein operons? o The mRNA synthesizes Ribosomal Proteins. o The Ribosomal Protein (r-protein) then binds to its mRNA and prevents further translation of the mRNA What is an example of how protein kinases affect the regulation of translation? o Globin Synthesis in Reticulocytes What is globin synthesis dependent on? o The phosphorylation of eIF2. Explain the mechanism of Protein Kinases in the Initiation of Translation. o eIF2 is stable when it is dephosphorylated o eIF2 is unstable with phosphorylated by KINASE o Heme inhibits Kinase o When Iron is present, Heme is Present o When Iron is present, Kinase is inhibited and eIF2 is stable and active o When Iron is not present, there will be no heme. The Kinase can phosphorylate eIF2 and deactivate it (it gets degraded) What is an Iron Responsive Element (IRE) o It is present in mRNA and it regulates the ferritin and transferrin receptor What is Ferritin? o It is an iron-storage protein that gets translated when there is excess iron What is Transferrin? o It is an iron-uptake protein that gets translated when there is iron deficiency During Iron Starvation, what will the cell translate? o Ferritin is not translated o TRANSFERRIN IS TRANSLATED During Excess Iron, what will the cell translate? o FERRITIN IS TRANSLATED The addition of iron removes the Binding Protein from the 5’ IRE – removes the “road-block” so it can translate o Transferrin is not translated. The addition of iron also removes the Binding Protein from the 3’ IRE – destabilizes the mRNA