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60 Cards in this Set
- Front
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gene expression regulation in proks |
-critical for cellular growth and env. respones -couped transc. trans limits points of control → mainly regulated by control of gene transcription Ex: ß-galactosidase induction: rate of mRNA synthesis parallels rate of protein production and levels repression: synthesis frops rapidly, enzyme levels slowly decline |
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prok transcription initiation of operons: ligand induction (relief of repression) |
repressor binds operator (adjacent to prok promoter) → no transcription inducer ligand bins repressor → repressor conformational change → decreased affinity for operator → falls off → transcription occurs *repressor has own gene, own promoter, different chromosomal location |
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lac operon |
inducible -Lac Z, Y, A (all three required for lac met=in operon) |
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lac operon in presence of lactose, no glucose |
-lactose → ß-glactosidase converts to allolactose → allolac binds lac repressor(1 per subunit of tetramer) → can't bind operator → transcription of lac operon -low glucose → high cAMP → binds CRP → CRP-cAMP binds promoter-proximal site → RNApol recruited → max transcription efficiency |
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CRP |
cAMP Receptor Protein -inducible activator of lac operon (requires cAMP to bind promoter proximal site) -regulates genes other than lac also -TF- helix-turn-helix family of DNABPs |
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lac operon in presence of lactose and glucose |
catabolite repression: glucose inhibits lac operon -high glucose = low cAMP levels → CRP can't bind promoter-proximal site -FB mechanism (glucose product of lac metabolsim) -regardless of lactose concentration if glucose is present transcription won't occur (cell has sufficient E) |
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lac operon in absence of lactose |
- no lactose → no allolactose → lac repressor dimers bind o1 and o2 → form tetramer → no transcription |
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two requirements for max transcription of lac operon |
1. absence of repressor (derepression) 2. presence of activator (true activation) **final transcription level depends on balance of these activities |
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Transcription factors |
-TF- helix-turn-helix family of DNABPs -regulate recruitment/activity of RNApol at promoter Exs: activators, repressors, CRP, lac repressor etc |
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CRP and lac repressor binding site |
perfect 2-fold symmetry around axis (diagsymmetry) → dimers bind CRP site upstream of promoter lac repressor down stream --o2 has less affinity, binds o1 first then helps recruit to o2 (cooperativity) |
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euk gene regulation |
1. transcriptional control: affects rate mRNA is produced by RNApol II *activation/repression by TFs *developmental reg *epigenetic control 2. post-transcriptional control: a. *RNA processing/alternative splicing/polyadenylation b. RNA transport and localization contorl 4. *translation control (protein concentration) 5. *mRNA degradation/stability control 6. *protein activity control |
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transcription rate and mRNA concentration in euks |
no correllation bw.n rates of transcription and concentration of m RNA in the cell → depends on mRNA stability |
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alternative splicing |
Exs: 1. optional exon 2. optional intron 3. mutually exclusive exons 4. internal splice site -results in 5 different protein isoforms possible → slighlty different biochemical activity -tissue-specific fashion or funvtion of developmental stage -how get high protein complexity w/ low gene number |
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alternate processing |
can include both alternate splict siite and alternate polyA site selection Ex: α -tropomyosin -key protein involved in mm. fibers and filamentous components of cytoskeleton -different isoforms in different cell types -has 3 poly A sites |
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ß+-thalassemia from intron 1 mutation |
results in two splice acceptor sites mutant site used 90% of time → 19 nts of intron included → frameshift mutation → premature stop → nonfunctional 10% normal |
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2. |
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3. lacZ gene coding sequence |
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2. high trp = binds repressor activating it → binds DNA and blocks transcription |
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lecture 1 |
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factors that determine concentration of mRNA in cytoplasm of a cell |
1. half-life 2. regulated degradation 3. translational regulation 4. RNA interference (via miRNAs/siRNAs) |
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mRNA half-life |
3'UTR sequence greatly affects - AUUUA -present in signaling molecules→ rapid turnover → short time for translation → few proteins produced → transient signal -inserted into 3'UTR of ß-globin → half life went from 10 to 1-2 hrs --inserted GC residues and had no effect |
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regulated mRNA degradation |
Ex: trasnferrin receptor synthesis -transport Fe into cell low Fe: -2 IRE-BP bind to 2 loops on 3'UTR → protected from degradtaion → increased translation of receptor high Fe: -Fe binds IRE-BP → can't bind 3' UTR → mRNA degraded → decreased receptor production |
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translation regulation |
Ex: Ferritin -Fe storage protein low Fe: -IRE-BP binds to loop in 5' UTR → translation prevented → Fe in cell made available high Fe: -Fe binds IRE-BP → can't bind 5' UTR → translatiion → increased Fe storage |
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RNA interference (RNAi) |
miRNA-RISC complex: -imperfect complementarity → targets many mRNAs → bind 3'UTR → translation prevented -perfect complementarity → binds anywhere on that mRNA → nucleases signaled to cut and degrade small-interfering RNA: (siRNAs) -externally supplied dsRNA in cytoplasm processed by dicer → siRNA duplex → unwinds so ss siRNA→ incorporated into RISC → specific mRNA degradation |
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miRNA |
-RNApol II transcribes -primary transcripts or exist w/n introns of other transcribed genes -processing: pri-miRNA in nuc form hairpin loops → Drosh clips → pre-miRNA → exportin 5 exports to cyto → Dicer clips → 21-23 bases long→ ds miRNA duplex → unwind and 1 strand incorporated into RISC complex (RNA-Induced Silencing Complex) **over/under expression → cancer |
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small-interfering RNA: (siRNAs) potential! |
externally supplied → suggested therapeutic approach to down regulate over expressed genes -Ex: treat cancer by degrading oncogene -treat other dominantly acting diseases |
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Euk genes and their transcriptional control elements |
-range from simple to highly complex: --yeast (bacteria-like): contain Upstream Activator Sequence (UAS), TATA, 1 exon no introns --mammalian: lots of enhancers (spacing doesn't matter), promoter proximal elements (spacing matters), introns and exons -often numerous, compound (bind many TFs), and variable locations (far from protein coding sequence, within sequence, downstream etc) |
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cell type, tissue and developmental stage specific gene expression |
-dependent upon match of approprate TFs and the regulatory elements they bind → enhancers critical control elements for this |
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what actually constitutes a gene |
the coding sequence + all of its transcription factor-binding regulatory elements |
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enhancers |
complex version of promoter proximal elements that bind TFs and interact w/ promoter sequences via DNA looping -variable locations (-50 kb + to +50 kb +) |
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transcriptional initiation complexes |
-critical to competent initation complex assembly: DNA-binding TFs interact via specific protein-protein contacts w/ other DNA binding TFs and mediator → "conversation" --requires DNA looping components: -basal transcription complex (basal TFs), mediator, Regulatory DNA binding TFs -bind gene regulatory sequences (HRE, enhancers, promoter proximal Es, core promoter etc) |
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mediator |
co-activator or co-repressor -non-DNA binding TF -integrates infor from different locations on DNA by interacting w/ other TFs |
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gene organization and developmental regulation at ß-globin locus |
-clustered on chromosome 11= 5 distinct ß-globin-like genes: in order of chromosomal and developmental appearance 1. Є (embryonic) 2. γG (fetal) 3. γ A (fetal) 4. 𝛿 (starts right before birth, accumulates to 2% in adults, irrelevant) 5. ß (adult) -as each gene is upregulated earlier expressed genes are downregulated -each gene has own regulatory elements → contributes to but not solely responsible for developmental activation and represion |
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complex interactions at ß-globin gene regulatory elements |
-combination of specific and general TFs bind at regulatory control elements → tissue-specific or developmental stage-specific expressoin Ex: GATA-1 : hematopoietic-specific factor EKLF: erythroid-specific factor (only expressed in RBCs) -TF mutation or DNA binding site mut → lower or no transc → genetic disease |
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promoter mutations in ß-globin gene |
-core promoter: TBP binding site -promoter-prox Es: EKLF binding site → ß-thalassemia (these SNPs confirm importance of EKLF and TBP interactions at this locus) |
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mutations that lead to complete regulatory failure at ß-globin locus: Locus control region (LCR) |
-far upstream enhancer → deletion → γ𝛿ß-thalassemia -enhancer controls overall expression and developmental gene switching -contains many TF binding sites -open chromatin only in cells of erythroid lineage → only controls in these cells where appropriate TFs can bind (highly condensed in nonRBCs) |
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local chromatin structure and TFs |
can only gain access and bind to enhancers that are open |
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DNA-binding TFs structure |
-euks and proks -modular = several functionally independent domains (each encoded in own exon?) -domains include: 1. DNA binding domain (DBD)- several families 2. Transactivation domain (TAD) - interacts w/ co-activtors, TFIIs and other TFs to form pre-initiation complex 3. Ligand binding sites- not all, exs: steroid hormone receptors, lac repressor, CRP 4. dimerization region- not all, allows homodimers or heterodimers 5. Nuclear Localization Sequence (NLS) - euks only 6. Repression domains- (present instead of TADs) 7. inhibitor binding sites? |
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Steroid hormone receptor |
N-TAD-DBD-dimerization sites-NLS-inhibitor binding sites-LBD-C |
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DNA-binding Domains (DBDs) |
1. Zinc fingers 2. leucine zipper 3. helix-turn-helix 4. helix-loop-helix *others known -α-helix inserted into major groove (sometimes minor-TBP_ -sequence specific interacction via H-bonding bw/n aa side chains and DNA bases/bps -homodimerization/heterodimerization of factors w/n a family→ different DNA sequence specificites (ex myc family factors) |
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Glucocorticoid receptor (GR) |
-Zn-finger binds as homodimer -recognition sequence: minor groove gap bw/n dyad symmetrical binding sites (A/G)GAACANNNTGTTC(T/C) |
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tissue-specific gene expression |
driven by particular combos of TFs -some TFs tissue specific -some TFs general → unique combo → tissue-specificity |
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transactivation domains (TAD) |
-mediates interactions bw/n TF and other protein complexes (co-activators, TAFs etc) -critical for transcriptional activation (*TAF = TBP-associated factor; TFIID=TBP + TAFs) |
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Transcriptional Repressor Domains (TRDs) |
repressor version of TADs |
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TFs and ligand binding |
Negative regulation: bound repressor → no transc 1. ligand binds repressor → transc. on 2. ligand binds repressor → repressor active → binds DNA → transc off Positive regulation: bound activator → transc 1. ligand binds → activator can't bind → transc off 2. ligand binds → activator active → binds DNA → transc on |
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activation of glucocorticoid steroid hormone receptor (GR) TF |
in cyto: no cortisol: HSP bound to GR→ can't enter nucleus cortisol: cortisol binds GR → conformational change → HSP cant bind → NLS, DBD, TAD revealed and forms dimer → enters nucleus → binds GRE on DNA→ transcription on *other TFs can be activated by modifying aa residues (phosphorylation, meth etc) |
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signal transduction and control of cell growth and proliferation |
overview: signaling molecule → receptor → intracellular transducers → secondary messengers → TFs → transc of cell cycle control proteins (ex cyclins) -pathwyas help control progression through cell cycle (CDK/cyclins help regulate progression, checkpoints) -mutations → overactivation of positive regulators (proto-oncs) or reduce/eliinate negative regulators (tumor-suppressors) → loss of cell cycle control → unregulated proliferation |
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proto-onc → onc |
1. radiation/chemical carcinogen -mutation in coding region → hyper -mutation in promotor → excessive expression 2. gene rearrangement -coding region swaps place w/ gene under strong promoter/enhancer -fused w/ another protein → over expressed/hyper 3. gene amplification -multiple copies of proto-onc each w/ own promoter → over exp |
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c-Myc and c-Fos |
-onc -helix-turn-helix TF -as proto-onc: indued transiently in cells that receive GF stimulation → transient cyclin activation → G1 → S and DNA synthesis |
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Burkitt's lymphoma |
-chromosomal translocation 8, 14 -myc (8) under control of highly active immunoglobulin gene heavy chain enhancer (CH) → constant myc expression in lymphocytes (gain of function)→ lymphoma |
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c-Myc and Rb |
myc activated → Rb inactivated → E2F active → transcription of cyclins → progression from G1 → S -mutated c-Myc → constant signal for cell proliferation |
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Rb |
tumor-suppressor -transcription co-repressor → binds E2F (a TF) → no cyclin A/E transcription → inhibits progression into S -myc → p16 releases cyclinD/CDK4 → phosphorylation of Rb → can't bind E2F → cyclins produced -LOF → constitutive activation of S phase genes *signal upstream doesn't matter |
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DNA in Euks |
-present as chromatin in nuclei → tightly folded and mostly inaccessible DNA w/ histones -fundamental packaging unit of all euk DNA: nucleosome |
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nucleosome |
-disc-shaped protein core: 8 highly basic histone proteins (positive) --2 x H2A --2 x H2B --2 x H3 --2 x H4 -2 turns of DNA around (acidic, negative) -histone tails: --flexible → no role in nucleosome formation --mediate internucleosomal packaging: many positively charged lys → allows interaction w/ adjacent nucleosomes (which are overed in (-) DNA) |
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condensed vs decondensed chromatin |
condensed: 30 nm chromatin structure -transcriptional repression → RNApol and TFs can't access. -ground state decondensed: chromatin acccessible |
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decondensation and recondensation of chromatin |
*reversible rxns -Histone Acetylases (HATS): acetylate (via AcetylCoA) lys on jistone tails → neutralized → no interaction bw/n neighboring nucleosomes --co-activators (TFs that are not DNA BP) --acetyl Lys → recognition sites for other proteins → further decondese → transcription -Histone Deacetylases (HDACs): allow charge-charge interaction to occur again --co-repressors (TFs that are not DNA BP) --major drug target |
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histone code (epigenetics) |
-histone aas can be chemically altered by large variety (100+) of post-translational modifications (PTMs) -predominantly acetlyation of lys -PTMs read by specific domains in various proteins → binding → co-activation/repression via further alteration of chromatin structure |
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histone code examples (on H3) |
1. methylation of lys 9 → heterochromatin 2. methyl lys 4, acetyl lys 9 → gene expression 3. phos ser 10, acetyl lys 14 → gene expressoin 4. methy; ;ys 27 → Hox genes silences, X chrom inactivation |
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decondensation of chromatin |
-many co-activators -some chemically modify histones -some recognize and bind specific histone mods → brind new enzyme activiies (move nucleosome, weakend DNA histone interaction, displace histones via ATP hysrolysis = chromatin remodeling) |
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cis vs trans elements |
cis: DNA regulatory elements trans: Transcription factors |