Gene Regulation

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gene expression regulation in proks

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-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)

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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

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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

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-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

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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

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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

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- 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

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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

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-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

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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

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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

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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

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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

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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

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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

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1. half-life

2. regulated degradation

3. translational regulation

4. RNA interference (via miRNAs/siRNAs)

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mRNA half-life

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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

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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

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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)

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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

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-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!

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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

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-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

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-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

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

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enhancers

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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

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-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

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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

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-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

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-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

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-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)

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-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

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can only gain access and bind to enhancers that are open

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DNA-binding TFs structure

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-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

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

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DNA-binding Domains (DBDs)

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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)

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-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

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driven by particular combos of TFs

-some TFs tissue specific

-some TFs general

→ unique combo → tissue-specificity

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transactivation domains (TAD)

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-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)

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repressor version of TADs

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TFs and ligand binding

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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

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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

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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

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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

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-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

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-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

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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

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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

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-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

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-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

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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

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*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)

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-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)

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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

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-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

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cis: DNA regulatory elements

trans: Transcription factors