Eukaryotic Gene Expression (Midterm 3 Lesson 4)

t/f: eukaryotic gene expression is more complex than prokaryotic

true

why is eukaryotic gene expression more complex than prokaryotic

• multiple organs

  • lots of diff organs/tissues that need certain things to be active

• have transcription in nucleus then translation in cytoplasm

• have chromatin (w DNA wrapped around histones)

t/f: eukaryotes organize their genes into operons. explain why or why not

false

what specifically about the transcription process in eukaryotes makes it more complicated than in prokaryotes

their control of transcription

• many steps can be regulated to control the amount of active gene product in eukaryotes as opposed to prokaryotes

how many diff RNA polymerases transcribe diff types of genes in eukaryotes

3-5

what are protein-encoding (structural) genes transcribed by

RNA polymerase II

what is a DNA sequence upstream of a gene that binds proteins that modulate the transcription of that gene called

a cis-acting factor

• factor that modulates the regulates a gene that is on the SAME DNA MOLECULE AS IT

• dna sequences in general are usually cis-acting factors while proteins are trans-acting

  • dna sequences can’t migrate to other DNA sequences but proteins can

what do trans-acting proteins bind to

cis acting regulatory regions

what are transcription factors

sequence-specific DNA binding proteins

• bind to promoters and/or enhancers

• recruit other proteins to influence transcription

what are the 3 types of transcription factors

• basal factors

• activators

• repressors

what are the major cis-acting regulatory elements

• promoters

• enhancers

where are promoters located, what are they composed of, what do they bind to, and what can they do on their own

• close to gene’s coding region (around 30bps upstream of start site), its a conserved sequence of TATA boxes (sections high in As and Ts)

• binds RNA polymerase and basal factors

• maintain a basal level of transcription on its own

what are basal factors, what are some examples, and what do they do? what is their mechanism of action

they maintain a basal level of transcription when binding to promoters of protein-coding genes

• proteins that are essential for the initiation of transcription in eukaryotic cells.

  • called "basal" because they are required for basic transcription to occur—i.e., they are needed for RNA polymerase II to begin transcribing most genes, even without any additional regulatory proteins

explain ordered binding at the promotor of eukaryotic cells

eg the TFIID complex → TFIID is one of the general (basal) transcription factors required for transcription by RNA polymerase II

• ordered binding at the promoter (occurs in this order)

  1. TATA box-binding protein (TBP) binds to TATA box (TBP = one basal factor)

  2. TCP-associated factors (TAFs) bind to TBP (TAF = basal factor)

  3. RNA pol 2 binds to TAFs

• TFIID = TBP + multiple TAFs

• TFIID is a basal factor

note: is only rna pol 2 and basal factors are bound to promoter, the gene maintains a “basal level of transcription” when this happens

what is a basal level of expression and what type of studies show this

the minimal level of transcription that occurs in the absence of activators and repressors

• in vivo studies show basal levels of rna

what do in vitro/in vivo studies show about basal levels of transcription (no activators/repressors)? explain

• in vitro (in test tube) → shows basal levels for most eukaryotic genes is high

• in vivo (in acc organism) → shows basal levels for most eukaryotic genes is much lower

  • bc of histones in cells → chromatin has DNA molecules wrapped around histones to form nucleosomes → makes it harder to transcribe

  • in vitro studies usually j have raw dna in it so it can go faster

what causes the basal level of expression in the absence of both activators and repressors

dna of eukaryotes is packaged into chromatin by the wrapping of dna around histone proteins to form units of nucleosomes → makes it harder to transcribe dna when it is wound like that (proteins for transcription have a really hard time binding bc dna is so tightlyt wound around histones)

NOTE: BASAL FACTORS are things that help rna pol bind to promoter

what are proteins that bind directly to the promoter and are necessary for rna pol 2 binding and transcription initiation?

basal factors (eg tata binding protein)

what are some methods gene expression is controlled by

• repressors and activators

  • repressors can recruit co-repressors (or not) and activators can recruit co-activators (or not)

• chromatin structure

  • acetylation of histones (turns transcription on)

  • methylation of histones (can turn transcription off or on)

    • depends on which AAs, # of methyl groups

• dna methylation of promoter (turns transcription off)

what are some diffs in how transcription is “turned off” in prokaryotes and eukaryotes

Bacteria:

• transcription is actively turned off by repressor proteins binding to the DNA

Eukaryotes:

• transcription is passively minimized due to packaging structure of chromatin

  • passively lowered bc wound so tightly around

• transcription rates depend on remodelling of chromatin structure and subsequent interactions w transcription factors

note: still have repressors in eukaryotes but transcription is passively minimized bc of histones ; repressors can LATER provide more specificity but are not main method of action

what are some other things besides rna pol that bind to promoters in prokaryotes and eukaryotes

prokaryotes:

• sigma factor (binds and guides rna pol)

  • sigma factors are a type of basal factor

eukaryotes:

• more/diff proteins

  • TAF

  • TBP

    • other proteins that bind to promoter and bind to rna pol

in both cases there are basal factors that bind to promotor in addition to rna pol

what is the basal factor for prokaryotes

sigma factor

what is the basal factor in eukaryotes

TBP TAF

what is the second main cis-acting regulatory element in eukaryotes

enhancers

where are enhancers located

• can be really close or really far away from the core promoter (even > 10,000bp away)

• can be upstream or downstream of a gene (either5’ or 3’ to the transcription start site)

  • still functional when moved to diff positions relative to promoter

even if you move it it will STILL HAVE THE SAME FUNCTION

• mostly bc dna can loop around itself and make it so enhancer and promoter are close tg regardless of where on the dna the enhancer is located

what is the purpose of enhancers

bind transcription factors (activators specifically) that inc the rate of transcription of a gene

• pull in transcription factors close to the promotor region so they bind more

what are the 2 domains activators have? what do they do

1. DNA binding domain (binds to DNA)

2. transcription activation domains (bind to basal factors or co-activators)

3. possible third domain → dimerization domain

what are activators

transcriptional activators that bind to specific enhancers and speed up the rate of transcription

• stabilize things bound to promoter so their action can be sped up

note: can also bind to repressors and inhibit them depending on the type of activator

what are the 2 ways activators can work in euks

1. can recruit co-activators to unwrap DNA of promoter region for transcription

• coactivators displace nucleosomes → they clear out the histones and free up the dna

  • allows basal factors and rna pol to bind

  • bc eukaryotic dna is wrapped around histones a lot of their regulation has to do w modifying the histones

2. can recruit basal factors and pol 2 and stabilize the interaction

• dna loops around so enhancer and promoter are across from each other

note: is on enhancer for both mechanisms of action

do basal factors bind to both the promotor and enhancer

• promotor → yes → needs basal factors to bind to directly allow rna pol to bind

• enhancer → no → gets bound to by activators or sometimes repressors

what are these all examples of

• basal factors

• activators

• repressors

different types of transcription factors

what do mediator proteins do

facilitate interaction between the activator and other proteins that are bound to the promotor

what are repressors

proteins that bind to selected sets of genes at sites known as silencers and thus slow transcription

where do activators act

right on proteins that are bound to the promoter (doing so inc rate of transcription by stabilizing their connection to promoter)

• able to so so bc dna folds to bring enhancer and promoter close tg

how do distant enhancers interact w genes?

distant regulatory elements can be brought closer tg by chromatin looping

• base of the loops held tg by proteins

  • CTCF

what is CTCF

• a zinc finger protein → holds loops of dna tg that bring enhancer and promotor closer tg

what prevents an enhancer from interacting w the wrong promotor

an insulator

what are insulators and what do they do

• regions of dna that bind proteins (eg CTCF) that facilitate dna looping

t/f: enhancers for promotors have to be on the same loop

true → it is cis-acting

explain what common motifs are in regards to transcription factors

recurring structural patterns (or domains) found in transcription factors that allow them to bind specific DNA sequences and/or interact with other proteins

• there are common motifs that are located on diff transcription factors (proteins that regulate gene expression)

• eg zink finger

  • each “finger” on these motifs have a helix that fits int he major groove of dna

  • the AAs adjacent to the dna determine which dna sequences are recognized

specific zink finger will recognize a specific dna sequence

explain the dimerization domain on some transcription factors

many eukaryotic activators need to form dimers (2 molecules bound tg) to fx

• happens via the dimerization domain

what is a common example of a dimerization domain on transcription factors

• dimers can come tg to form a leucine zipper

• each molecule in dimer has leucine residues that bind tg w lucine residues on the other protein

what is an activator that works in the lac operon in prokaryotes

CRP (a positive regulator) of lac transcription

• is active when bound to cAMP, its inducer (instead of a coactivator in eukaryotes)

• binds to dna and inc efficiency of rna pol binding and activitiy

what are the equivalents to coactivators and corepressors in prokaryotes

prokaryotes have inducers instead

what are some diff ways coactivators can act

• bind allosterically to activators and change their conformation

• changing histone proteins

• interact w proteins at promoter

t/f: coactivators are all proteins

false → estrogens, testosterone, cortisol are all co-activators

explain how some steroid hormones can work as co-activators

testosterone, estrogen and cortisol can all bind to specific transcription factors (activators specifically) → activators in this case are also considered “hormone receptors”

• change the receptor (activator’s) shape (allosteric control)

• allows them to bind to another molecule and form a dimer

• allows them to bind to an enhancer

• enhancer loops towards promotor region

• inc transcription rate of target gene

note: j one example of what an activator could do (many diff examples)

which molecules are present during basal levels of transcription

• basal factors (TBP and TAF)

• rna pol

  • all bound to promoter

• nothing bound to enhancer

what do transcriptional repressors do

lower transcriptional activity

what can repressors bind to

• enhancers (specific DNA region) ; competition or recruitment of co-repressors)

• silencers (specific dna regions)

• activator (protein ; quenching)

what are the 2 kinds of repressors

direct and indirect repressors

what do direct repressors do

• recruit corepressors that either…

  • directly prevent rna pol 2 complex (rna pol 2 + TAF + TBP) from binding to promoter

  • close chromatin (around histones)

what are the 2 ways indirect repressors act

• competition

• quenching

explain how indirect repressors that act through competition work

repressor binds to enhancer and competes w activator

• if repressor binds to enhancer, activator can not

explain how direct repressors that act through quenching work

repressor binds to activator and prevents it from functioning (does NOT bind directly to dna like indirect repressors that act through competition)

what do direct repressors do

• actively make sure histones are there and dna is tightly bound

• stoping proteins from being able to bind from promoter

what are 2 ways direct repressors can work

1. recruit corepressors that directly prevent rna pol 2 complex (basal factors specifically) from binding to promoter

• can to bind basal complex and prevent them from binding to promoter bc they’re binding to corepressor instead

2. recruit corepressors that close chromatin

   • induce wrapping of promoter dna around histones → directly inhibits transcription initiation

t/f: repressors and enhancer can both bind to enhancer and silencer regions of dna

false → both can bind to enhancers, only repressors can bind to silencers

what is chromatin remodelling

when the structure of chromatin is altered to control gene expression (cant transcribe gene that is tightly wound around histones)

• can either make easier or harder

what is histone acetylation

type of chromosome remodelling

• acetyl groups get added to histones directly

  • normal histones have naturally + charges

  • acetylation adds acetyl groups that have - charges

    • dna also has - charged groups (ie phosphate groups)

    • makes it easier to unwind dna and for proteins to bind to promoter

    • inc rate of transcription

what does histone acetyl transferase do

catalyzes rxn where acetyl group is added to histones → type of coactivators

• inc rate of transcription

what is histone methylation? explain it and provide a specific example

type of chromosome remodelling

• methyl groups are added to histones

• has diff influences depending on where its added

• mostly results in histones being held closer tg than they normally would be

  • dec rate of transcription

can be relatively permanent

• eg can create heterochromatin (a tightly packed form of chromatin)

  • in which genes are essentially silenced

  • basically j permanently turns them off

note: specific methylation can also decrease affinity of histones for dna (co-activators would add in this case)

what does histone methyltransferase (HMT) do

catalyzes addition of methyl groups to histones

• is a co-repressor (dec rate of transcription)

what is heterochromatin

• tightly-packed form of chromatin (in which genes are essentially silenced)

• created by specific types of methylation

which structure on chromosomes contain large regions of heterochromatin

barr bodies → in inactivated x chroms are entirely made of heterochromatin

what do dark bands on chromosomes usually indicate

regions with heterochromatin (even more tightly-packed dna) → dna in these regions is not transcribed

what are dna methylation patterns

another way to silence genes

• many promoters have blocks of CG-rich sequences called CpG islands (p stands for phosphodiester bonds) → can be methylated and turned off

  • the acc promoter in the dna sequence itself is methylated → turns it off

what are epigenetics

changes in gene expression NOT related to sequence changes in the dna

• eg if you have identical genomes, if both have diff modifications to them (even if they don’t change the sequence itself), they could still have diff phenotypes

  • eg through diff methylation patterns

    • if something is cloned, it will not look the same

what are heritable epigenetic markers

epigenetic markers that can be passed down to offspring

what is gene imprinting

specific epigenetic marks (like dna methylation) are placed on certain genes during gamete formation, depending on whether the gamete is sperm or egg → happens naturally, don’t know exactly why → most likely just gene regulation

• maintained in mitosis so all cells in body have only one allele expressed

  • other is silenced

t/f: diet can also influence methylation patterns

true → and they can be inherited

• end up w diff phenotypes despite having identical alleles

explain how diff mice w same genotype (heterozygous for coat colour → results in yellow coat) can have different coat colours with the exact same alleles. what experiment was used to text this

remember: yellow gene is lethal when homozygous

2 regions on yellow gene

P1 region: normal gene promoter, weak

• methylated = only P2 is expressed, very yellow

P2: second, very strong promoter, can be methylated

• methylated = only P1 is expressed, barely yellow

fed 2 pops of hetero mice (yellow) → one w methylated diet, other with regular…

• methylated diet (P2 off) were brown, slim, low risk of cancer and diabetes

• no methylation (P2 on) were yellow, obese, high risk of cancer and diabetes)