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60 Cards in this Set

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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

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

lac operon

inducible


-Lac Z, Y, A (all three required for lac met=in operon)



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

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

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)

lac operon in absence of lactose

- no lactose → no allolactose → lac repressor dimers bind o1 and o2 → form tetramer → no transcription

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

Transcription factors

-TF- helix-turn-helix family of DNABPs


-regulate recruitment/activity of RNApol at promoter


Exs: activators, repressors, CRP, lac repressor etc

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)

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

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

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

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

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

2.



3. lacZ gene coding sequence





2. high trp = binds repressor activating it → binds DNA and blocks transcription

lecture 1

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)

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

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

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

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

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

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

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)



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

what actually constitutes a gene

the coding sequence + all of its transcription factor-binding regulatory elements

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 +)

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)

mediator

co-activator or co-repressor


-non-DNA binding TF


-integrates infor from different locations on DNA by interacting w/ other TFs



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



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

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)

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)



local chromatin structure and TFs

can only gain access and bind to enhancers that are open

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?



Steroid hormone receptor

N-TAD-DBD-dimerization sites-NLS-inhibitor binding sites-LBD-C

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)

Glucocorticoid receptor (GR)

-Zn-finger binds as homodimer


-recognition sequence: minor groove gap bw/n dyad symmetrical binding sites


(A/G)GAACANNNTGTTC(T/C)

tissue-specific gene expression

driven by particular combos of TFs


-some TFs tissue specific


-some TFs general


→ unique combo → tissue-specificity

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)



Transcriptional Repressor Domains (TRDs)

repressor version of TADs

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

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)

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

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

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

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

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

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



DNA in Euks

-present as chromatin in nuclei → tightly folded and mostly inaccessible DNA w/ histones


-fundamental packaging unit of all euk DNA: nucleosome

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)



condensed vs decondensed chromatin

condensed: 30 nm chromatin structure


-transcriptional repression → RNApol and TFs can't access.


-ground state




decondensed: chromatin acccessible



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

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

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

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)

cis vs trans elements

cis: DNA regulatory elements


trans: Transcription factors