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MCB 2050 Midterm Questions With Complete Solutions What happens if the 'silencer' is deleted? The adjacent locus is transcribed Which loci are constantly repressed in yeast? The mating type loci HML and HMR What is the function of Rap1? The repressor; it binds to the repressed loci and recruits SIR proteins What is the function of SIR2? It deacetylates the histones with HMR and HML What happens after to the histones that are deacetylated by SIR2? They bind tighter to DNA and form heterochromatin to prevent the association of transcriptional activators to promoters What is necessary for positional gene repression? Histone hypo-acetylation What do Rap1 and SIR proteins bind to? The telomeres What is the function of SIR proteins in the telomeres? They spread in the sub-telomeric regions and cover them with condensed hypo-acetylated nucleosomes
What occurs in a lysine --> arginine substitution in histone tails? Arg retains its positive charge and cannot be acetylated so the mutant maintains permanent gene repression at the telomeres What occurs in a lysine --> glutamine substitution in histone tails? Gln has a neutral charge and cannot be deacetylated so the mutant maintains permanent gene activation at the telomeres (abolish gene repression) What is the role of SIR3/4? Co-repressors that bring in SIR What do repressors and activators NOT directly bind? DNA What is different about promoter-dependent repression/activation vs. position-dependent? The position of the genes is not the critical factor, the promoter is because the co-repressors/activators do not spread, they stay localized at the regulatory elements What is Ume6? A transcription repressor in yeast that is recruited and brings the Sin3/Rpd3 complex What is the Sin3/Rpd3 complex? A co-repressor in yeast What is Rpd3?
What is the Mediator a major 'bridging' factor between?
- transcriptional activators
- GTFs
- histone modifiers
- chromatin remodeling factors What is a major disrupter of chromatin and bound transcription factors? DNA replication What is epigenetic 'memory'? Cells differentiate and select the gene they express then the cells divide multiple times, but continue to express the same genes after each cell division How do cells remember which genes to express? Mainly through reconstitution of the same chromatin structure after the passage of the replication fork What are epigenetic marks? Histone post-translational modifications What is DNA methylation associated with? Heterochromatin and repressed genes What is histone acetylation associated with? Active genes and euchromatin What is histone deacetylation associated with?
Gene repression and heterochromatin What is histone methylation associated with? Both gene activation and repression, depending on the specific modifications What are the important sites of modification of methylated lysine and their effect on transcription?
- H3 (K4) Me3 in promoter region: activation
- H3 (K4) Me1 in enhancers: activation
- H3 (K9, K27) Me2/3: repression Unmethylated CpG islands recruit proteins that methylate a specific position on histones: Histone 3-Lysine-4 (H3-K4) What is H3-K4 recognized as? An epigenetic mark that dictates gene expression What is the role of PRC2? Maintains repression by associating with target loci and continuously methylating H3-K What is the role of PRC1? Recognizes H3-K27-Me and compacts chromatin What is the role of trithorax complexes? Oppose repression by polycomb complexes by methylating H at Lys 4 and maintaining the activating modification during chromosome replication
tRNAs and rRNAs, that have to be processed before they can be functional and exported from the nucleus Where does splicing occur for short primary transcripts with few introns and long genes with multiple introns?
- short: follows cleavage and polyadenylation
- large: before transcription of the gene is complete (directly out of nascent RNA) What are two examples of alternative splicing?
- alternative splicing in neurons: perception of sound
- sex determination in Drosophilia What molecules does Western blot study? Proteins What molecules does Northern blot study? RNA What molecules does Southern blot study? DNA Which of the following is a function of a promoter? Select all that apply.
- regulate the rate of replication
- regulate the efficiency of translation
- provide a binding site for transcription factors
- position the +1 site
- provide a binding site for the ribosome
- provide a binding site for transcription factors
- position the +1 site Reverse transcriptase is an enzyme which catalyzes: The synthesis of cDNA from an RNA template Quantitative PCR (qPCR) is ultimately used to? Quantify the amount of target DNA in the original sample Quantitative Reverse Transcription PCR (qRT - PCR) is ultimately used to? Amplify and quantify target mRNA in the original sample cDNA has the same sequence as its template i) ________ but consists of ii) ________. i) mRNA; ii) dNTPs A protein has the following target residue sequence which is recognized by a kinase protein; R-X-X-B-R-S -X-X-B. Which amino acid residue within this sequence will be phosphorylated by the kinase? Serine i)________ add ii) __________ groups to target proteins, while a iii)________ remove them. i) Kinases; ii) phosphate; iii) phosphatases CDKs are activated when they are i)____________ their respective cyclin molecules and function by ii)______________ other molecules.
and 4 and the removal of the stop codon, continuing production of Sxl Males do not repress splicing together exons 2 and 3 and produce the stop codon that prevents further production of Sxl What occurs with Tra in male and female Drosophilia? In females, Sxl represses splicing together exons 1 and 2; instead it splices together exons 1 and 3 removing the stop codon and producing Tra protein Males do not produce Sxl so repression of splicing together 1 and 2 does not occur and the stop codon is produced which prevents production of Tra protein What is the result of Tra production or inhibition in male and female Drosophilia? In females, Tra cooperatively binds with Rbp1 and Tra 2 to splice together exons 3 and 4 and polyadenylating at the 3' end of exon 4 of Dsx In males, without Tra and Tra2 or Rbp1, exon 3 and 5 are spliced together and a different Dsx protein is produced What is the function of Sxl? An intron splicing silencer (inhibitor) What is the function of Tra? An intron splicing activator What does the female isoform of Dsx contain? A transcriptional activation domain
What does the male isoform of Dsx contain? A transcriptional repression domain How do miRNAs regulate gene expression? They IMPERFECTLY bind to target mRNA, which leads to repression of translation (might only be a brief pause) Where do miRNAs generally bind? 3'-UTR of mRNA How do siRNAs silence gene expression? They PERFECTLY bind to target mRNA, interfering with the stability of the mRNA and causing it's rapid degradation by a mechanism called RNA interference What is Drosha? An endonuclease that removes the 5' cap and poly A tail in the double-stranded RNA to convert pri-mi-R-1-1 miRNA into pre- miRNA What is the function of Exportin? Transports the processed miRNA to the cytoplasm through the nuclear pore complex What is the function of Dicer? It cuts the double-stranded RNA into small pieces and removes the hairpins in the double-stranded RNA What is Argonaut?
proteins/second messengers
- signal transductions leads to activation of effector proteins What does activation of effector proteins cause?
- short-term responses
- long-term responses
- termination (or down-regulation) from negative feedback
- removal of the extracellular signal from the receptor What are examples of hydrophilic hormones?
- small molecules: epinephrine, acetylcholine
- peptides: glucagon
- proteins: insulin, growth hormones What does Dex affect? It activates the glucocorticoid receptor by bypassing the chaperones and allowing the receptor to enter the nucleus What is endocrine? It affects target cells some distance from the site of synthesis (use of blood stream for transport) What is paracrine? It affects target cells in close proximity (no blood stream involvement) What is autocrine? It affects target receptors on itself What is signaling by plasma-membrane-attached proteins?
The hormone is not secreted, it sits on the cell surface and the target receptor is on an adjacent cell What is the function of kinases? Removing a phosphate from ATP and using it to phosphorylate target proteins What is the function of phosphatases? Oppose the affects of kinases by removing the phosphate How can kinase/phosphatase activity be regulated?
- phosphorylation
- binding to other proteins
- binding of second messenger molecules What are the two types of kinases in animal cells?
- tyrosine kinase
- serine/threonine kinase Which hormones activate G-proteins? Hydrophilic hormones What are the immediate recipient of hydrophilic hormones that activates G-proteins? G-protein coupled receptors (GPCR) How is G-protein activity regulated? The exchange of GDP to GTP What switches G-protein 'on'?
They carry and amplify the signals from receptors (G-proteins) to effector molecules What are examples of 2 water-soluble secondary messengers and what do they activate?
- cAMP: activates PKA
- cGMP: activates PKG and opens cation channels in rod cells What are examples of 2 lipid-soluble secondary messengers and what do they activate?
- DAG: activates PKC
- IP3: opens Ca2+ channels in the endoplasmic reticulum Which receptor subunits cause stimulation and inhibition of cAMP?
- Gas-coupled receptors stimulate cAMP
- Gai-coupled receptors inhibit cAMP Adenylyl cyclase synthesizes the diffusible second messenger____________ from ____________ cAMP; ATP What is the amplification pathway of epinephrine? An epinephrine molecule binds to a GPCR, which activates several molecules of adenylyl cyclase, each molecule catalyzes the synthesis of several cAMP molecules, 2 molecules of cAMP activate 1 PKA but each PKA phosphorylates multiple target proteins What are G-proteins?
On-Off switches for intracellular signaling pathways by activating or inactivating ion channels or effector enzymes that generate second messenger molecules What some examples of cellular activities that GPCR signaling pathways regulate?
- metabolism
- cell division
- gene expression What is the structure of GPCRs?
- 7 hydrophobic transmembrane a-helical regions (H1-H7)
- ligand-binding site located in a groove in the middle of the plane of the membrane What is the mechanisms of GPCR signaling?
- Hormone binds receptor and induces a conformational change
- Activated receptor can now bind to Ga subunit
- Activated receptor causes conformational change in Ga, which triggers dissociation of GDP
- GEF provides a GTP which binds to Ga and triggers dissociation of Ga from the receptor and Gβγ subunits
- The hormone dissociates from the receptor and Ga binds and activates the effector
- GTP is hydrolyzed to GDP, causing Ga to dissociate from the effector and reassociate with Gβγ What technique is used to reveal the kinetics of G-protein activation?
Induces conformational changes that leads to the kinase's activation What does activation of dimerized RTKs lead to? Cytoplasmic domain autophosphorylation What mediates signaling by different members of the epidermal growth factor family of hormones? 4 HER RTKs How is sensitivity to many peptides hormones reduced? Endocytosis of receptor-hormone complexes by lysosomes to reduce the number of cell-surface receptors What is the MAP kinase pathway?
- RTK activation leads to the autophosphorylation of the RTK intracellular domain
- The phosphorylated intracellular domain interacts with GRB and Sos through SH2 and SH
- Sos activates Ras, which binds GDP and converts it to GTP
- Ras activates Raf (MAPKKK)
- Raf phosphorylates MEK (MAPKK)
- MEK phosphorylates ERK (MAPK)
- ERK translocates to the nucleus What links RTK to Ras? GRB2 and Sos What does Sos act as?
GEF; promotes dissociation of GDP from Ras so GTP can bind/activate Ras What domains does GRB2 have? 2 SH3 domains and 1 SH2 domain SH3 binds? Proline-rich motifs What stabilizes Raf in its inactive conformation? Binding to 14-3-3 protein dimer What causes dissociation of the 14-3-3 protein dimer from Raf? Binding of the Raf N-terminal regulatory domain to Raf-GTP What is meant by 'G-proteins have intrinsic GTPase activity'? Often the protein that binds them acts as the GTPase activation protein, which causes hydrolysis of GTP to GDP What happens after ERK (MAPK) is activated?
- it activates P90-RSK which moves into the nucleus and phosphorylates a specific Ser in the SRF transcription factor
- translocates to the nucleus and phosphorylates specific Ser in transcription factor TCF, which is already bound to c-fos These actions both stimulate stimulate transcription of c-fos What is the mechanism of action of PI-3 kinase pathways?
- PI-3 kinase is recruited to activated RTK by its SH2 domain and a phosphorylated tyrosine on RTK
- PI-3 phosphorylates PIP/PIP2, which generates membrane-
