Regulation of Gene Expression
What is a very unique characterisitc of the Lac Operon?
It exhibits both positive and negative regulation
what is GATA?
A zinc finger containing transcription factor that is involved in cardiac muscle development
Repressors have subunits with _____
HDAC - Histone deacetylase activity
Which type of chromatin is highly condensed
Heterochromatin
Negative regulation in regulation by a _____
Repressor
True or False Regulation at the translational level is more important for eukaryotes than for prokaryotes
True
True or False: Trp is a co-repressor for its own biosynthesis and thus exhibits negative feedback
True
Co-repressors might also bind histone _____ which compact the chromatin
methyl transferase Methylation = heterochromatin formation (inactive transcription)
What is needed for RNA Polymerase 2 to bind to a promoter?
1) Basal transcription factors (TBP, TFIIB) needed at every Pol II promotor 2) DNA-binding transactivators which bind to enhancers and facilitate transcription (Protein-DNA) 3) Coactivators which act indirectly: bridge between DNA-binding transactivators and complex composed of Pol II and the general (or basal) transcription factors. (protein-protein domains) TFIID coactivator activity is needed for many genes (is complexed with TBP and TAFs) Mediator coactivator is required for both basal and regulated transcription at promotors used by RNA Pol II. Binds to the C-terminus of the large subunit of RNA Pol II and stimulates phosphorylation of that region by TFIIH
What are the 3 classes of regulatory proteins?
1) Specificity factors: alter specificity of RNA polymerase for promotor -Sigma factor in bacteria -TBP protein in eukaryotes 2)Repressors: impede access of RNA polymerase to the promotor (mostly in bacteria). Regulation by a repressor is called negative regulation 3) Activators: enhance RNA polymerase - promotor interaction. Regulation by an activator is called positive regulation
Give a summary of the process you just learned (G Protein/Epinephrine)
1. Signal (epinephrine) binds to GPCR making it active 2. Stimulatory subunit of the GPCR activates adenylyl cyclase 3. Adenylyl cyclase promotes synthesis of cAMP 4. cAMP binds the regulatory subunits of PKA, releasing catalytic subunits 5. Catalytic subunits move to nucleus and phosphorylate CREB 6. Phosphorylated CREB binds to CRE 7. CBP binds to CREB 8. Gene transcription is initiated
What are the two classes of enzymes that play a role in chromatin remodelling?
1. enzymes that covalently modify the core histones of nucleosomes: Histone acetyl transferases (HATs) and Histone Deacetylates (HDACs) Acetylation status of amino-terminal domains of histone H3 and H4 determines the affinity between nucleosome and DNA acetylation removes + on Lys of histone!! 2. Enzymes that use ATP to remodel nucleosomes on the DNA: SWI/SNF complexes that actively move or displace nucleosomes Create hypersensitive sites in the DNA and stimulate binding of transcription factors
The nucleotide sequence of the 5'-end of trp mRNA includes a short Open Reading Frame (ORF) that encodes a ____ amino acid peptide containing two Trp residues and has an untranslated attenuator region
14
When TATA box is knocked out, promoter activity drops to what level?
38%
What is an example of an iron sensor?
4Fe-4S cluster in aconitase
TRP Repressor can reduce expression by _____ X Attenuation can reduce expression by _____ X Together they can reduce by _______X
70 10 700
2.) Since 2 Trp must be bound to the repressor in order for it to bind to the operator, what kind of molecule is it? A.) Co-repressor B.) Inducer C.) Co-Activator D.) Who cares!!!
A
When Heme is present at low or no concentration: A. Heme kinase is active, phosphorylating eIF2, and no translation of globin occurs B. Heme kinase is inactive, phosphorylating eIF2, and translation of globin occurs C. Heme kinase is active, no phosphorylation occurs, and no translation of globin occurs. D. Heme kinase is inactive, no phosphorylation occurs, and translation of globin occurs
A
Describe the process of negative regulation
A repressor bound to the dna sequence operator, inhibits transcription A molecular signal binds, causing repressor to dissociate from DNA Can also be when a molecular signal binds, causing repressor to bind to DNA to inhibit transcription Positive regulation is the same except when bound it Facilitates transcription
What removes the + charge on Lysine of histones?
Acetylation
Regulatory systems that act in trans?
Activator or repressor proteins reach the point of action by diffusion: these are acting in trans
What protein is responsible for maintaining the glucocorticoid receptor in its inactive form? A. CBP B. Hsp90 C. PKA D. SWI/SNF
B
Product of Lac Z Gene?
Beta Galactosidase
So what are the different scenarios of positive regulation and transcription involving the different signal molecules?
Bound Activator Transcription Co-Activator bound to Activator Activator binds DNA Transcription
1.) When a molecule binds to a protein that is bound to the promoter region causing it to fall off and transcription results; what kind of regulation is taking place and what kind of signal molecule is it? A.) + Regulation; co-activator B.) + Regulation; Inducer C.) - Regulation; Inducer D.) - Regulation; Co-activator
C
3.) Under which conditions will you see gene products from the Lac Operon?? A.) Allalactose bound to activator; cAMP bound to repressor B.) Allalactose bound to CAP; cAMP bound to activator C.) Allalactose bound to repressor; cAMP bound CAP D.) Lactose bound to repressor, cAMP cound to CAP
C
In the case of Iron Regulation, when Fe is high: A. Transferrin is made from the 5' IRE B. Transferrin is made from the 3' IRE C. Ferritin is made from the 5' IRE D. Ferritin is made from the 3' IRE
C
Which one of the following DNA to protein interactions is found in both prokaryotes and eukaryotes? A. Leucine Zipper B. Helix Loop Helix C. Helix Turn Helix D. Zinc Fingers
C
Deficiency/mutation of _____ can result in a complex genetic disorder
CBP (rubenstein-taybi syndrome)
What is an operon?
Cluster of genes involved in the same biochemical pathway Polycistronic mRNA is the product of an operon Operon includes the promoter region which contains next to a binding site for RNA polymerase (sigma subunit) also a binding site for an activator and for a repressor. The repressor binding sequence has a special name: operators
What do you know about the Tyrptophan (TRP) Operon?
Consists of promoter region and 5 genes involved in tryptophan biosynthesis In promoter a 15 basepair region is located which is recognized by a protein. As this protein inhibits further expression it is called a repressor. However, the repressor protein binds only efficiently to the operator if it has bound two Trp molecules-(co-repressors) Repressor (transcribed from a gene elsewhere in the E.coli genome) is always present in 20 copies per cell (constitutive expression)
What do you know about the Lac Operon?
Consists of structural genes involved in lactose hydrolysis, a gene coding for a constitutively expressed repressor and an operator sequence for a repressor and another sequence for an activator (CRP site) Repressor is inactivated by binding to allolactose (lactose with an alpha1,6 bond).-Allalactose is INDUCER Lactose operon can only work when it is properly activated by CRP (cAMP Responsive Protein) also known as CAP (Catabolite Activator Protein) which can only bind to the CRP site when it has bound cAMP.-(Co-activator of CAP)
5.) Which statement is true about chromatin remodeling? A.) HAT uses ATP to move nucleosome down activates transcription B.) HDACs acetylates Lys residues of histone activate transcription C.) SWI/SNF complexes use ATP to move nucleosome deactivate transcription D.) HAT acetylates Lys on Histone activates transcription
D
In Breast Cancer, certain estrogen pathways are needed for progression. Tamoxifen is a drug that inhibits the use of these estrogen pathways by: A. Binding to the estrogen ligand inhibiting activation of the estrogen receptor. B. Blocking the activity of the co-repressor to the estrogen receptor C. Changing the conformation of the estrogen ligand so that it cannot bind to the receptor. D. Acting as an estrogen analog, and blocking the activity of the co-activator of the estrogen receptor.
D
Which one of the following proteins acts as a co-repressor, that binds histone methyltransferase ending in chromatin compaction? A. HAT B. Cytosolic aconitase C. Tamoxifen D. HDAC
D
Summarize what you've learned about Protein-Protein and Dna-Protein interactions
DNA/PROTEIN Helix turn Helix (prok) Helical region interacts with DNA= recognition Helix. Rich in NQEKR Helix turn Helix (euk) Transcription factors 60aa DOMAIN=HOMEODOMAIN Corresponding gene=HOMEOBOX Zinc Fingers (euk) Zn does NOT directly interact w/ DNA Need multiple fingers Confers DNA binding stability Ex: GATA-cardiac muscle development HMG: Box motiff- bring distal promoters closer, allow for DNA bending!!! Protein/Protein Leucine Zipper: Leucine residues -> hydrophobic regions ->loosen aliphatic side chains -> form dimers ->coil of alpha helices Heterodimers allow for making more transcription factors Helix Loop Helix 50aa protein dimer formation Mediated by a stretch of basic aa (bHLH) MyoD- TF involved in muscle differentiation containing bHLH
What are the roles of enhancers, dna looping and insulators?
Enhancers may be thousands of basepairs away from the transcription initiation site and make contact with the initiator sites through transactivators. DNA looping allows for interaction between distant DNA sites (HMG proteins)- DNA bending Insulators limit the action range of enhancers: might bind to nuclear matrix giving looped domains- separate domains
Describe Gene Activation by Steroid Hormone
Enters cell by diffusion Binds hormone receptor in the cytoplasm Binding causes the receptor to translocate to the nucleus In the nucleus the receptor acts as a transcription factor and binds to the hormone response element (HRE) of the DNA, so here it becomes a DNA-binding transactivator Binding of two adjacent receptor molecules leads to formation of a dimer, which activates transcription
Using epinephrine as an example discuss protein phosphorylation and gene activation
Epinephrine activates G-protein coupled receptors Activated G-protein binds to and activates adenylyl cyclase giving increasing cAMP level cAMP binds to regulatory subunit of protein kinase A (PKA) and the released PKA catalytic subunits move to the nucleus PKA catalytic subunits phosphorylate the cAMP-Response Element Binding Protein (CREB) in the nucleus P-CREB binds to cAMP response elements in the promotors of target genes and binds then the coactivater CREB Binding Protein (CBP): histone acetylation and chromatin remodelling facilitating transcription
What type of Chromatin has Acetylated Histones (HATs)?
Euchromatin
True or False Heme-Kinase is the active form
False - inactive Kinase is active form
What is IRE? How does its effect change based on if its at 5' or 3'
Ferritin and the transferrin receptor are reciprocally regulated by the same DNA element: Iron Responsive Element (IRE) In the IRE-binding protein the site for iron binding and for DNA binding overlap. Excess iron causes dissociation of Binding Protein from the IRE Binding of IRE-Binding protein to the 5` IRE blocks translation of ferritin mRNA Binding of the IRE-Binding protein to the 3` IRE's stabilizes the transferrin receptor mRNA
Product of Lac Y Gene?
Galactose Permease
Principles of Gene Expression
Gene products rise and fall in response to molecular signals: regulated gene expression Inducible genes- increase expression Repressible genes- decrease expression Gene products stay more or less constant: constitutive gene expression House keeping genes- for cell function Levels of control: transcriptional and translational
What is the role of protein kinases in the initiation of translation?
Globin synthesis in reticulocytes is dependent on phosphorylation state of eIF2 (binds to initiator tRNA), that is that unphosphorylated eIF2 is the active variant The abundant presence of heme promotes initiation of translation by direct inhibition of the kinase In the Presence of Heme, Your Heme Kinase will be INACTIVE, since translation needs the UNPHOSPHORYLATED eIF2 to actively initiate translation
Give and example of how gene expression is coordinated in Eukaryotes
Glucocorticoid receptor is a transcription factor upon binding of the hormone cortisol. Multiple genes are activated by cortisol Such a receptor may be the last missing factor for a number of genes to be transcribed. So we get coordinated gene expression in response to increased levels of circulating hormone.
What is glucose's role in the regulation of the Lac Operon?
Glucose lowers intracellular cAMP levels by inhibiting adenylyl cyclase, causing also cAMP dissociation from CAP (glucose effect, catabolite repression) Glucose is the preferential carbon source for E coli, but it may also utilize lactose in the absence of glucose. Lactose turns on lac operon immediately. When both lactose and glucose are present, glucose is used preferentially (glucose effect) So both glucose and lactose affect the lactose operon
What does Histone Acetyltransferase do? and what is its effect?
HATs catalyze transfer of acetyl groups from Acetyl Coenzyme A to various Lys and Arg residues in amino terminal regions of the histone core proteins. Effect of acetylation of histone tails: prevent chromatin from folding in compact structures, thus maintains the active euchromatic state (loosening DNA-histone interaction) Acetylation of histone tails means also: increased access of specific DNA regions to chromatin remodelling complexes proteins
What does HMG stand for? and what is its significance?
HMG name is derived from High Mobility Group (relates to their discovery, but not to their function) Three alpha-helical regions interact with DNA DNA bending allows for interaction with other transcription factors on nearby sites: architectural factors SRY gene responsible for male sexual differentiation contains a HMG box
Which type of chromatin is highly methylated even at promoters
Heterochromatin
Which type of chromatin is transcriptionally inactive?
Heterochromatin
Attenuation of mRNA occurs when there are high/low levels of TRP?
High
What is the role of the promoter in expression?
In the absence of regulatory sequences, differences in promotor sequences can account for 1000-fold difference in mRNA product: Major determinant for expression of house keeping genes Deviations from consensus sequence usually decrease promotor function
What is Allalacotse?
Inducer - causes dissociation of repressor from Dna allowing transcription
What is transcription attenuation?
Inhibition of gene expression by premature termination of transcription Attenuation is a mode of transcription regulation for operons of amino acid biosynthesis Upstream of the structural genes in the operon lies a short Open Reading Frame (ORF) for about 14 amino acids A relatively large number of the codons in the short ORF code for the amino acid, whose biosynthesis is directed by the operon (Negative Feedback) Remember: in prokaryotes transcription and translation are tightly coupled: translation starts before transcription is completed
Product of Lac I Gene?
Lac repressor
Lactose can oly work when it is properly activated by _____ which is also known as ______ which can only bind when it has bound ______ which is a _____
Lactose operon can only work when it is properly activated by CRP (cAMP Responsive Protein) also known as CAP (Catabolite Activator Protein) which can only bind to the CRP site when it has bound cAMP.-(Co-activator of CAP)
Discuss protein-protein interaction domains in transcription factors
Leucine zipper: Leucine-rich regions in two proteins interact to form the dimer (coiled coil of alpha-helices). Adjacent to the zipper region is a basic aa segment that interacts with DNA (total is called bZIP domain). enhanced by formation of heterodimers Fos and Jun, proteins involved in cell proliferation, contain both bZIP domains Helix-Loop-Helix region of 50 aa is involved in protein dimer formation. DNA binding is mediated by an adjacent short stretch of basic aa (bHLH) MyoD, a transcription factor involved in muscle differentiation contains a bHLH domain
What are DNase hypersensitive sites in chromosomal DNA?
Marks regions in chromosomes that are devoid of nucleosomes and allow for binding of transcription factors MAKE PRODUCT It are regions of 100-200 bp in length within 1000 bp upstream of the transcription initiation site, but can also be located elsewhere. DNase hypersensitive sites differ from tissue to tissue for selected genes: cell type specific gene expression Please note: DNase is only a labtool here TEST FOR STATE OF DNA!!
What is the role of DNA methylation in gene expression?
Methylation in eukaryotic DNA is limited to Cytosine bases in CpG dinucleotides (methylation in prokaryotes centers on A in GATC sequences) Methylated DNA is associated with gene-silencing. (x-inactivation) Demethylation of DNA with activation MeCP proteins bind to methylated CpG dinucleotides MeCP2 recruits HDAC activity removing acetyl groups from histones and inducing chromatin remodelling MeCP2 then aids methylation of H3K9 HP1 (Heterochromatin Protein 1) binds to Methylated H3K9 and to itself, condensing the chromatin
What are the general features of Eukaryotic Transcription Regulation?
Mostly positive regulation Promotors are only active in the presence of regulatory proteins At least 5 regulatory sites for of any eukaryotic gene which may be thousands of base pairs away from the transcription start site and even downstream of the coding sequence Access to eukaryotic promotors is restricted by the structure of the chromatin (HISTONES) Heterochromatin: no gene expression (condensed) Euchromatin: gene expression in particular regions (Loose)
What are 3 ways to measure gene expression?
Northern Blot RT/PCR DNA Microarry
Describe the different scenarios involved when glucose and lactose are involved in the Lac Operon
Note: Glucose is preferred over Lactose
Regulatory systems that act in cis. What does this mean?
Regulatory elements like promotors and enhancers are on the same chromosome as the transcribed gene: they are acting in cis
So what are the different scenarios of negative regulation and transcription involving the different signal molecules?
Repressor bound Operator Inhibits Transcription Inducer repressor falls off Transcription Co-repressor repressor binds operator NO Transcription
Transitional feedback occurs in ribosomal protein operons..explain
Ribosomal proteins act as the inhibitors of continued mRNA synthesis when there are adverse growth conditions (synthesis of rRNA stops, ribosomal protein accumulates)
Specific sigma factors guides RNA polymerase to specific promoter sequences. What sigma factor is for heat shock proteins? What sigma factor is for most proteins?
Sigma 32 for heat shock Sigma 70 for most proteins
Mutations in the HOXD13 GENE cause?
Synpolydactyl
Summarize what you've learned about Protein-Protein and Dna-Protein interactions
TF/DNA - helix-turn-helix- Prokaryotes and Eukaryotes Homeodomain (Eukaryotic) - zinc fingers-need more than one. Protein/Protein Leucine zippers (heterodimers ) Helix-loop-helix
What is Tamoxifen and its use?
Tamoxifen is an estrogen analog and binds in pocket normally occupied by estrogen Tamoxifen binding distorts the structure of the estrogen receptor and thereby blocks the binding between the coactivator to the estrogen receptor Some breast tumors rely on estrogen-mediated pathways for growth So Tamoxifen can be used for anti-cancer
Discuss attenuator region and hairpins
The attenuator region consists of the ORF for the leader peptide and three regions which can engage in two different hairpins (proximal and distal downstream of the ORF coding for the leader peptide) When there is enough of the amino acid the operon codes for, then the leader peptide is readily synthesized and the distal hairpin is favored, resulting in premature termination of the transcript When there is not enough of the amino acid the operon codes for, the ribosome stalls at the sequence coding for the leader peptide and the proximal hairpin is favored, resulting in complete transcription of the operon
What is the attenuator in attenuation?
The leader region is a regulatory region located between the operator and the initiaton codon of the first gene. The non-translated region between the short ORF and the start of the structural genes is called the attenuator. In the attenuator there are three regions which can engage in hairpin formation (2,3,4). Only one hairpin formed of two regions is possible at any time The 5' located hairpin does not inhibit RNA polymerase, while the 3' located hairpin does.
Nuclear Hormone Receptors have 2 conserved domains
The nuclear hormone receptors have 2 conserved domains: DNA binding domain (2 zinc fingers) and a ligand binding domain (ligand =hormone) DNA binding sites are called hormone responsive elements (HREs) on DNA There are about 50 nuclear hormone receptors in human
Signal Molecules can be referred to as _______ depending on their roles in positive/negative regulation
The signal molecule is called the CO-REPRESSOR when binding to the repressor promotes binding to the operator and is called INDUCER when binding to the repressor causes dissociation from the operator. The signal molecule is called CO-ACTIVATOR when binding to the activator induces binding to the promotor
What is Transferrin and when is it used?
The transferrin receptor is involved in the uptake or transferrin-bound iron by cells. The latter protein is particularly needed in case of iron deficiency (Iron-Starved) while in the transferrin receptor mRNA a number of IRE's are located in the 3'-untranslated region
What is the purpose of deletion mapping?
To find regulatory sequences
What is Homeodomain?
Transcription factors in human development contain a helix-turn helix domain of 60 aa that is called homeodomain. The corresponding segment in the gene is called a homeobox
True or False: Only Prokaryotes have operons?
True
True/False: Co-repressors lead to transcriptional repression
True
Many Human Transcription Factors contain Zinc Fingers. What do you know about these "fingers"
Zinc 2+ ion held by 4 Cys or 2 Cys and 2His (2 aa from an alpha-helical region and 2 aa from beta-sheet region) Domain is about 20 aa long Zinc does not directly interact with DNA Interaction between a single Zn finger and DNA is weak Multiple zinc fingers substantially enhance binding by interacting simultaneously with the DNA
Dimeric R (RR) activates what?
activates genes which have a hormonal responsive element HRE element in their promotor through chromatin remodelling coactivators
What is Ferritin and when is it used?
an iron-storage protein required when there is an excess of (dietary) iron Ferritin mRNA has the IRE in the 5`-untranslated region of the mRNA
Describe the DNA binding dna domain
apha-helical region inserted in the major groove, 60 - 90 amino acids long
Describe Dna-Protein Interaction
involves Hydrogen bonding and hydrophobic interactions (methyl group of Thymidine) Amino acids involved in DNA binding are asparagine, glutamine, glutamate, arginine, lysine Affinity of a particular transcription factor for its target sequence in DNA is a million fold higher compared to other DNA sequences As yet not possible to predict to which DNA sequence a given transcription factor will bind DNA target site usually palindromic repeat to which 2 subunits of a regulatory sequence bind cooperatively. Doubling of the contact sites squares the association constant!
Glucocorticoid receptor (R) is normally bound by _____ What happens when a Glucocorticoid binds instead?
the HSP 90 protein and is inactive Binding of hormone to R releases it from HSP90, induces dimerisation of R, exposes a nuclear localisation signal, and RR moves to the nucleus
How do many bacterial regulatory proteins interact with DNA?
through helix-turn-helix motifs in the protein. The helical region interacting with the DNA is called the recognition helix (rich in N,Q,E,K or R) Helix-Turn-Helix domain (20 aa) of the Lac repressor interacts with DNA (helical regions of 7-9 aa)
