BIMM 100 Final Exam Notes PDF

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Zhang 1

Monday (2/25) Gene regulation ● Epithelial cells + neurons ○ Have the same DNA but make different  mRNA → different  proteins → different  functions ○ Same cell type but different environmental conditions (ie. same DNA but with/without hormones → make different mRNA → different proteins → different functions) ● Eukaryotes vs. bacteria ○ Eukaryotes

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■ Each gene has its own promoter (many TSSs + promoters) ■ Transcribed separately; translated separately Bacteria: ■ 1 TSS + same promoter (eg. 3 genes are under the same promoter + share a TSS) Transcribed together into 1 mRNA; translated  separately into proteins A, B & C Operon: a cluster of genes under a single promoter AUG at the beginning of gene A; UGA at the end of gene A Ribosomal binding site (RBI): a small piece of specific RNA (due to no 5’ cap + 3’ poly A tail); beginning of gene

■ ■ ■ ■

(before AUG) ● Ribosome first finds the RBI, then finds the first start codon → falls off when it finds the stop codon ● ●

Same thing applies to genes B + C → each contains its own RBI, start codon & stop codon → translated separately (many ribosomes working simultaneously)

Sugar metabolism in bacteria ○ First eats all the glucose (glycolysis → energy), then eats other sugars (lactose) ○ ○

Only eats glucose → will only express the enzymes to digest glucose Eats other sugars → expresses the enzymes to digest the other sugars ■ (1) lactose permease: an enzyme that moves lactose (large sugar) into the cell ■ (2) B-galactosidase: an enzyme that cuts lactose into glucose + galactose (then, digests glucose → glycolysis → energy)

Lac operon in bacteria ● iClicker question: the promoter region regulates the lac operon ● | lacZ | lacY  | lacA | ○ Lac Z protein product: B-galactosidase

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○ Lac Y protein product: lactose permease ○ Lac A  protein product: unknown Operon (organization) = efficient way of controlling gene expression ○ 1 promoter is responsible for an entire metabolic pathway ○ Repressed by glucose + activated by lactose | CAP | lac I |--------------------| CAP | CORE | operator | lacZ | lacY  | lacA | ○ CORE promoter (general functional element) recruits sigma factor (general TF) + polymerase ○ CAP site: activating functional element (recruits 2 C  AP → activates sigma factor + pol) ○ Operator: repressing  functional element (recruits TF lacI → represses sigma factor + pol) ○ CAP: makes CAP  I ○ lacI: makes lac ■ Glucose represses CAP  binding (activator) ● Represses cAMP (helps DNA bind to CAP) → activates Pol-II-CORE binding ■ Lactose represses lacI  binding (repressor) ● Represses lac I DNA-binding (binds to lacI + prevents DNA from binding) → activates Pol-II-CORE binding

Glucose (given)

Lactose (given)

CAP binding

lac I binding

Operon expression

+

–

–

+

No

–

+

+

–

Yes

–

–

+

+

No*

+

+

–

–

No (weak)

Zhang 2

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*repressor overrides activator 2 requirements for DNA expression: there is no glucose, there is lactose Regulated separately (glucose + lactose) ○ There is glucose → operon is off ○ There is only lactose → operon is on iClicker: no problem w/ CAP, problem w/ lac operon (or outside lac operon: problem w/ lac I gene)

Wednesday (2/27) Western blot ● Goal: to detect a specific  protein (protein of interest) ● Procedure: ○ Extract total protein from the cell → test tube ○ Run all protein (not charged) on gel; DNA + RNA are (–) charged → migrate on gel ○ Treat all protein w/ SDS (detergent) to charge → denatures protein + adds (–) charge

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○ Run all protein (charged) on gel → migrate on gel ○ Transfer to membrane → submerge in solution w/ antibody (binds  to target protein) ○ Antibody is tagged → will see a single  band Interpreting results: ○ Antibody = anti-B-galactosidase Glucose

+

–

Lactose

–

+ (band)

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Darker band = more protein (B-galactosidase)

Eukaryotic gene regulation ● Enhancers: far away upstream/downstream or inside introns; binds TFs → activate transcription ● PPEs (promoter proximal elements): binds TFs (can either activate or prevent transcription) ● CORE promoter (general promoter): binds TFII + Pol-II (1) enhancers ● Different from promoters (need to be close to/determines TSS & determines transcription direction) ○ (1) can function over a long distance ■ Folding → enhancer is physically close to promoter (physical vs. sequence distance) ○ (2) position/orientation-independent ■ Can put in any direction → still works (2) cis-regulatory elements (DNA) = enhancers, PPEs & CORE  promoter ● CORE promoter needs PPEs + enhancers to promote gene activation ○ PPEs + enhancers ■ Control the level of gene expression by recruiting TFs Control where (in which cell type) + w  hen (under which environmental conditions [eg. with/without hormone/sugar]) ■ Cell type/condition → determines TFs → determines cis-regulatory elements → controls gene expression TFs are different (determine  gene expression through the cis-regulatory elements) ■

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Every cell type has the same DNA + same enhancers + PPEs

(3) TFs (trans-regulatory elements) ● TFs are often modular (AD & DBD can function separately) ●

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O~O ○ O = activation/repression domain (AD) ○ O = DNA-binding domain (DBD) ○ ~ = linker sequence DBD binds → AD does work on TSS ○ DBD recognizes specific DNA sequence

Zhang 3

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■ Different TFs have different DBDs that recognize/bind to different DNA sequences DBD = specificity (AD = functionality) Friday (3/1)

GAL4 transcription activator is modular ● Procedure: ○ Use a reporter gene (binds GAL4) ○ Use deletion series on GAL4 (remove AD, DBD, linker → check binding + activity) ● Results: sometimes binding but no activity ● Conclusion: AD & DBD work independently Modular ● ●

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DBD (for gene 1) but AD (for gene 2) → can activate gene 1 b/c DBD is specific to gene 1 & AD is general (1) DBD ○ Binding specificity: depends on interaction w/ bases (sequence recognition) ○ Binding affinity: depends on interaction w/ phosphate, sugar & bases  (2) AD ○ TFII + Pol-II  recruitment via a mediator  (a large protein complex), itself, or other binding proteins ○ Chromatin regulation To activate transcription (not just binding): DBD + AD

Chromatin regulation ● Chromatin: DNA wrapped around histones (protein); histones   protect DNA & repress  transcription ● Nucleosome: ~150  bp of DNA wrapped around 8 histones; one of many repeated units of chromatin ●

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Chromatin states (for a specific genomic region, depending on cell type, environmental conditions, etc.) ○ Chromatin modifiers (acetylation/methylation): make chemical marks on histones to change chromatin state; slow ■ Euchromatin: open, active ■ Heterochromatin: closed, inactive (compact, tightly wrapped) ○ Chromatin remodeling complex (DNA helicase + ATP): modify euchromatin to expose CORE promoter; fast Not all genes have all 3 states (depends on the gene; some just have 1 state) Chromatin modifiers + remodeling complexes are recruited to specific DNA by TFs or DNA-binding proteins (how transition b/w states is regulated)

Monday (3/4) DNase protection assay – to determine the chromatin  state of a specific DNA region ● Procedure: DNA → test tube + DNase ● Cell type I (euchromatin state) ○ DNase can make cuts in between nucleosomes ○ Remove histones + BamHI digestion → shorter DNA ● Cell type II (heterochromatin state) ○ ○ ●

No cuts b/c nucleosomes are close together (DNase cannot make cuts) Remove histones + BamHI digestion → full-length DNA

Southern blot (visualization): filter membrane → fragment detection by probe hybridization 1

2

– – –

–

Interpretation: cell type I has shorter DNA (euchromatin state); cell type II has longer DNA (heterochromatin state) Chromatin modifications (heterochromatin  euchromatin) + modifiers  ● 8 histones (in 1 nucleosome): 2 H2A, 2 H2B, 2 H3, 2 H4 ○ K+ (K lysine): many outside the histones; (–) DNA can bind tightly ● Modifications: chemical marks added on specific lysines of histones ○ Modifiers: enzymes that add chemical marks on histones ● (1) histone acetylation/deacetylation: ○ Add acetyl groups to lysine → euchromatin state

Zhang 4

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(1) adding acetyl groups to lysine → loss of (+) on lysine → less DNA binding (2) adding acetyl groups to lysine → histones bind to proteins w/ bromodomains (often transcription activators)

Modifiers: ■ Histone acetylase (HAT): adds acetyl groups to histones → euchromatin ■ Histone deacetylase (HDAC): removes acetyl groups from histones → heterochromatin (2) histone methylation/demethylation: ○ Adding methyl groups to K4s (lysines) [t rimethylation] → euchromatin state ○ Adding methyl groups to K9s & K  27s (lysines) → heterochromatin state ■ Adding methyl groups → no change on charges ○

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■ Adding methyl groups → binds to proteins w/ chromodomains (either transcription activators or repressors) Modifiers:

■ Histone methyltransferase: adds methyl groups to histones (specific ones to specific lysines) ■ Histone demethylase: removes methyl groups from histones (specific ones to specific lysines) (3) heterochromatin formation + spreading: ○ Heterochromatin region = large genomic region (contains many genes) ○

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Heterochromatin spreading ■ Removing acetyl groups → Sir2 (a HDAC) binds better to histones w/o acetyl groups → removes acetyl groups from the next region of histones (chain rxn) ■ K9 methyltransferase binds → adding methyl groups to H3K9 → recruits HP1 (heterochromatin protein 1) → recruits more methyl transferases to neighboring histones (chain rxn) Boundary elements: DNA elements (cis-regulatory) that stop  heterochromatin spreading ■ (1) generate nucleosome-free regions ■ (2) recruits factors that repress chromatin modifiers

Wednesday (3/6) Chromatin immunoprecipitation (ChIP) [in vivo] ● Purpose: to determine whether  + where a specific protein (TF) binds to a specific DNA region ● ●

Similar to DNase footprinting (in vitro) Procedure: ○ (1) fixation (crosslinking): add formaldehyde to live cell → makes binding permanent ○ (2) isolation: isolate chromatins; sonication (shear DNA into pieces) ○ ○ ○

(3) immunoprecipitation: add antibodies to pull down TF of interest + its associated DNA ■ Antibody/TF/DNA complex precipitates @bottom of test tube (4) reverse crosslinking: DNA dissociates from TF (5) DNA analysis: w/ PCR ■ ■ ■ ■

Design many primer pairs (forward + reverse) + label primers Primers generate pieces of DNA Add template (DNA from test tube) to each test tube (contains a primer pair + its DNA) Run on gel → will see a band where TF binds to DNA

ChIP ● ● ● ●

Purpose: to determine the DNA  region that is acetylated/methylated HAT binds to TF  → adds acetyl groups to DNA that is immediately downstream Same procedure w/ different  antibody (binds to acetylated/methylated histone, not TF) Will see bands (after TF) that correspond to the DNA regions that are acetylated

Environment-dependent gene regulation ● ● ●

In the cell: RD–DBD–LBD (TF) No drug (glucocorticoid): LBD binds to inhibitor protein (anchors TF in the cytosol, will not move to nucleus) With drug (glucocorticoid): drug binds to LBD (releases inhibitor) → TF moves to the nucleus + binds DNA → RD (repression domain) recruits histone deacetylase (HDAC) → removes acetyl group from gene → represses gene expression (of inflammatory genes)

Tissue-specific gene regulation ● Cell types → tissue-specific TFs bind + activate enhancers (on the gene; cis-regulatory elements) → gene expression ○ Pax6 (gene) is present in every cell type but is only expressed in pancreas, eyes & brain ○

Pancreas-specific TF or brain-specific TF

Zhang 5

Friday (3/8) Alternative splicing ● Change splicing patterns in different cell types ● Result: an exon is spliced out → mature mRNA has 2 exons instead of 3 ● Mechanisms: ○ (1) Splicing silencers (pre-mRNA) recruit + bind splicing repressors (RNA sequences) → inhibits  recognition of 3’ + 5’ splice sites (ie. thinks exon 2 is part of intron) ■ Tissue 1 does not have the 2 repressors; tissue 2 has the 2 repressors ○ (2) Splicing enhancers (pre-mRNA) recruit + bind splicing activators (RNA sequences) → activates  recognition of weak splicing sites (ie. does not splice out an exon that would’ve been spliced out) ■ Without help (of splicing activators), spliceosome will not recognize the 2 sites ■ With help (of splicing activators), spliceosome will recognize the 2 sites ■ ■

Tissue 1 has the 2 splicing activators; tissue 2 does not have the 2 splicing activators RBD~SR (RBD = RNA-binding domain; SR = Ser/Arg-rich domain)

Alternative polyadenylation ● Add a polyA tail to a different location in different cell types Alternative promoters ● Different TFs in different cell types → recognize different promoters ● Result: different transcriptions...