MBB 331 midterm 3

Histones

Proteins that chromatin can fold around to make it more condensed

Nucleosomes

Chromatin wrapped around histones

2 nm DNA

Just the normal DNA helix

10 nm DNA

Beads on a string form of chromatin. Basically just chromatin wrapped around histones

30 nm DNA

Like 10nm but all the histones are squished closer together

Nucleosome structure

DNA wrapped around histone

Histone

Linker: DNA between nucleosomes (connecting them together)

How to determine nucleosomes exist

Digest the DNA (do dnase footprinting) and of there are pieces of increasing DNA around 200bp long, there are nucleosomes. Works because only the linker DNA will be digested only cutting between nucleosomes

RNA poly 1 product

rRNA

How much DNA per nucleosome

200 bp per nucleosome

Histone types used in forming nucleosomes (5)

H2A

H2B

H3

H4

H1

Assembly of nucleosome

H3 and H4 (2 of each) form a tetramer that binds to dsDNA

Two copies of H2A-H2B dimer are recruited to complete assembly of nucleosome

Histone tails

Tails that come out from the histones

Have a C terminal tail and an N terminal Tail

Composed of lysine and arginine. Can be modified by increasing or decreasing positive charge

Function is to interact with other nearby histones and can grab them bring nearby histones closer together condensing DNA

They are positively charged so they can interact with negatively charged DNA backbones

Acetylation of tails can make them less positively charged allowing for less interaction with DNA lessening condensation increasing transcription

H1 function

Leads to even more compaction of DNA by binding to the DNA on the histones

Increasing the amount of H1 increases the amount of compaction reducing transcription

Discovery of H1

Ran a western blot on differently condensed DNA. Basically just ran condensed DNA with H1 and without. H1 did not have any transcription leading to the discovery that it condensed DNA so much that transcription could not be performed on it

Hypersensitive regions

Control regions of actively transcribing genes are sensitive to DNase. Basically you can determine if a gene is active by using DNase on it and if it actually cuts something then it was active. This occurs because the DNA is usually covered in proteins that protect it but when it is being transcribed it will be unwound and unprotected. If run on a gel after all proteins are removed, you can compare with the same piece of DNA but not cut and if they are the same then it was not actively transcribed at that time. If different then it was because it was cut

Two classes of enzymes regulate the nucleosome arrangement

1. Chromatin remodeling complexes

Ejecting: Removing a nucleosome and returning to uncondensed state (Activates transcription)

Repositioning or replacing nucleosomes (Effect depends)

2. Histone modifying enzymes

Modifications of the N tails on the histones using the histone tail modification proteins

Histone tail modification proteins and which histone is modification occurring on

HATs

HDAs

HMT

HDM

All tail modifications occur on histone 4

HATs

Histone acetyltransferase. Adds acetyl group to histone tails: Increases transcription by decreasing condensation. Has bromodomains (allows them to bind to already acetylated histone tails and acetylate the nearby tails as well)

HDAs

Histone de acetyl transferase. Removes acetyl groups. Represses transcription by increasing condensation

HMT

Histone methyltransferase: Adds methyl groups to histone tails

HDM

Histone demethylase: Removes methyl groups from Histone tails. Results in more transcription becuase it will be less bulky

Bromodomains

Allows proteins involved in transcription to bind to acetylated histones. Basically just allows transcription of chromatin that is already exposed

Chromodomains

Allows proteins to bind to methylated histones

Histone modification inherited

Not encoded in the DNA but must be preserved during replication. The histones during replication will be preserved and histones will be added to the daughter strand at the same place a histone is found on the parent strand. Basically just ensures the daughter strand has the same histone sites as the parent.

SWI/SNF

These are the chromatin remodeling complex proteins.

They can eject or slide nucleosomes.

Ejecting nucleosomes increases transcription

Sliding nucleosomes can depend but can induce transcription by reveling promoter regions

Associates with histones using a bromodomain and activates transcription

Bacterial RNA polymerase vs eukaryotic RNA polymerase

They look very similar in their core units.

Eukaryotic has a mediator complex attached to it

RNA poly 2 product

mRNA

RNA poly 3 product

tRNA

How to isolate and study eRNA polymerase

Almost like chip sequencing but in this case they only care about the RNA polymerase and subunits

Basically tag an antibody onto the RNA polymerase. Because there's sequence homology between bacterial polymerase as e polyemrase, you can just put the antibody that would bind to bacterial and hope it binds to eukaryotic. Because the antibody is bound to one subunit and all twelve sub units are attached together, you can purify using that one antibody and get the whole RNA polymerase. Then you can just denature the RNA polymerase and then run the whole thing on a gel. They got 12 bands one for each subunit

TBP

TATA binding protein helps other things bind to the TATA box

Binds in the minor groove of the DNA

TAFs

TBP associated factors. Recognize promoter sequences and call TBP

RNA polymerase 1 preinitiation complex consists of

RNA pol 1

1 SL1 (selectivity factor)

2 UBFs (upstream binding factors) for high transcription

SL1 consists of

1 TBP, 3 TAFs

Location of RNA polymerase 3 promoters

Found within genes

tRNA promoter general transcription factor binding order

TFIIIC binds first

TFIIIB containing TBP binds after

rRNA promoter general transcription factor binding order

TFIIIA

TFIIIC

TFIIIB

RNAP 2 core promoter regions (4 of them)

TATA box is the promoter region. Located 26-31 bp upstream from transcription start site

BRE gene: located before the TATA box and is the TFIIB recognition element

DPE: Downstream promoter element

INR: Initiator sequence

RNAP2 core promoter general positions of elements from left to right (5 things)

BRE

TATA

INR

DPE

Any enhancer anywhere on the DNA strand

RNA pol 2 preinitiation complex consists of

General TFs

RNA pol 2

Promoter

Linker scanning mutagenesis to detect promoters

1. Break DNA and then ligate a linker into the DNA

2. Repeat for a bunch of the same strand of DNA but inserting the linker at different places near or in the promoter

3. So then you allow the gene to be expressed and measure how much of the protein is made

4. You can compare the amount of protein being made to the control

5. If there is nothing showing up on the gel then the linker was probably replacing the whole promoter (TATA box)

Prokaryotic vs Eukaryotic promoter regions

Prokaryotic has all enhancer elements nearby to promoter region, eukaryotic typically has them further away

TATA box in eukaryotes are located in the same place every single time for every gene and is the only consistent sequence

Eukaryotes require general transcription factors just for basal level of transcription

RNAP 2 transcription eukaryotic vs prokaryotic

RNAP 2 requires general transcription factors for basal transcription

Promoters can be controlled by DNA binding transcription activators. DNA binding proteins that act on DNA upstream of core promoters

Coactivators: Bind proteins for communication between RNAP, general TFs and activators

General transcription factors

TFIID (TBP of TFIID binds to TATA box)

TFIIB (Binds to BRE next to TBP)

TFIIF (Joins the complex of TFIID and B while holding RNA pol 2)

TFIIE (Joins complex)

TFIIH (Joins complex)

TFIIA (only used invivo unmasks TBP)

Called general transcription factors because they are used in nearly every single gene transcribed by RNA pol 2

What do activators bind to

Enhancers

Set of transcription factors to form preinitiation complex in order

TFIID + TFIIA

TFIIB

TFIIF + RNA polymerase 2

TFIIE

TFIIH

TFIID importance

Without TFIID nothing else binds

TFIID complex comprises of

TBP and 11 TAFs

TFIIF 2 subunits

Larger, RAP74. Like helicase

Smaller, RAP 38, similar to the sigma factor, finds the promoter region and binds tightly

Transcription initiation steps

TFIID recognizes the TATA box

TBP in the TFIID binds the TATA box

TFIIB binds to BRE which is next to the TATA box

TFIIF join the complex and bring RNAP 2 along with it

TFIIE joins

TFFIH binds to TFIIE. TFIIH phosphorylates the CTD tail on RNAP 2 to signal the start of transcription

RNAP changes shape and loses ability to bind all the other general TFs which causes them to unbind and frees RNAP to move away from promoter to start transcription

CTD tail

Basically a chain of amino acids attached to RNAP that can be phosphorylated or dephosphorylated to control transcription

Proteins that interact with CTD tail

TFIID: Interacts with the tail to initiate the formation of phosphodiester bonds

TFIIH: Phosphorylates the CTD tail

How to boost transcription

By binding activators to enhancer sequences in the DNA

Mediator complex

Bound to RNAP.

Activation: Increases the efficiency of assembly of preinitiation complex through directly interacting with activators

Down regulation: Can also reduce the efficiency of transcription

Basically works by bridging the looped back strand of DNA with activators on the enhancer sequences to the RNA polymerase 2

Enhancers

Basically just a sequence of DNA that activators can bind to. The sequence of DNA is not directional and can face any way and the activator will still bind. They basically just allow for the binding of more proteins in close proximity to replication machinery to increase rate of transcription

Enhancer orientation and position

Not dependent on any of them. It can face any way and be in any position as long as it's close enough to the TSS when the DNA loops around

Enhancer binding structure and how it interacts with the transcription machinery

Enhancer elements located pretty far away from TSS so how does it help transcription machinery bind? DNA will curve around the transcription start site in a way that allows the enhancer sequences to be above the TSS. Activator proteins will bind to the enhancer sites and interact with the mediator complex which is bound to the TSS basically forming a loop bridged by the transcription machinery.

Termination methods in eukaryotes

Allosteric model: Kinda like hairpin termination, a conformational change of the elongation complex causes the transcription machinery to fall off

Torpedo model: Like rho dependent termination, something cuts the RNA transcript after it is completed, exonuclease binds to the side of the transcript still in RNA polymerase and degrades it until it reaches the RNA polymerase which causes it to fall off

DNA melting proteins involved in transcription

RNA pol 2

TFIIH

Chip part of chip chip and chip sequencing

A way of isolating strands of DNA that have a protein bound to them and only those sequences of DNA. Do the same thing in DNase foot printing to cut up the DNA everywhere but the proteins and then attach antibodies to the proteins on the DNA and use those to separate that DNA from the rest. Wash off the proteins and you will be left with only the DNA that was bound to proteins

Chip sequencing vs Chip CHip

Chip sequencing is very specific and sequences the DNA

Chip Chip it's not very specific. Maybe it is just binding some similar sequence to the isolated DNA

Transcriptional ground state

restrictive in eukaryotes, no transcription

Reasons why transcriptional ground state is restricted in eukaryotes (4)

Condensed DNA makes DNA difficult to access

Activation is needed for transcription of almost all genes

Combinatorial control: is used when different combinations of TFs can be used to regulate multiple genes in different ways

Unique mechanisms of gene regulation in eukaryotes

Condensed DNA makes DNA difficult to access (two types of DNA)

Closed DNA: No acetyl groups attached to the histones. No transcription as it is condensed and inaccessible

Open DNA: Acetyl groups attached to histones allows for DNA to be uncondensed and accessible to transcription machinery

Acetylation of histones effect on DNA

Causes histones to separate from each other releasing DNA making it more susceptible to transcription

Activation is needed for transcription of almost all genes (What kind of things are needed for activation

All 3 RNA polymerases do not have good affinity for promoter regions

Multiple activators are needed to start transcription

Multiple regulators are used for every gene so they need to be removed before transcription can start. Needed because the promoter region of TATA could appear anywhere in eukaryote DNA (very common)

Combinatorial control

is used when different combinations of TFs can be used to regulate multiple genes in different ways

Basically like lac operon and crp and CAMP

Example found below

What are insulators in gene regulation?

DNA sequences bound by insulator proteins (e.g., CTCF) to block activators from affecting neighboring genes by ensuring activators activate the correct gene.

What is the function of enhanceosomes in gene regulation?

Cooperating activators that form stable nucleoproteins to enhance transcription by tightly folding DNA.

How does gene silencing occur? (After transcription has already occurred)

After RNA transcript formation, it is degraded to prevent its function. Only occurs when a transcript is made and is not needed

What is imprinting in gene regulation?

Methylation at specific DNA sites prevents certain protein binding, thereby regulating transcription initiation or termination.

Used to deactivate one of the chromosomes or certain alleles to deactivate an extra chromosome to avoid double the gene product

What is dosage compensation in gene expression?

It balances gene expression from sex chromosomes by reducing duplicate chromosome gene expression in females to avoid double the gene dosage.

How do steroid hormones regulate gene expression?

They bind to nuclear receptors to modulate gene expression levels.

Heterochromatin inaccessible DNA

Basically just DNA that is wound around histones and is inaccessible. Generally found at telomeres because they don't have any coding DNA so they don't have a reason to be unwound

Regulatory elements location in eukaryotes

Regulators are found distant from core promoters because there's so many of them not all of them can fit near the promoter sequence.

How to get around the distant regulatory elements

When DNA is being transcribed, the DNA bends around the RNA polymerase coming into contact with the mediator sequence. Basically the DNA bending around the RNA polymerase allows regulatory sequences way downstream to stay in contact with RNA polymerase

Histone code

A successive sequence of events directed by histone modification to drive gene transcription. Basically goes from wound up DNA to unwound DNA that can be accessed by transcription machinery (Slide 14 don't have to memorize steps) Every gene has different modifications done to histones but the goal is the same for all genes

Activator for gal

Gal4p is the activator for all gal genes

Gal coding genes

GAL80

GAL1

GAL3

GAL2

Gal80p

Inhibitor for Gal4p. Works by binding to Gal4p while it is bound to the DNA so it can't interact with the RNA polymerase anymore

Gal3p

Senses when galactose is present and if it is then inhibits Gal80p. Works by forming a complex with ATP and then binds to Gal80p allowing the whole complex to bind to the DNA. Then Gal4p can then bind to the Gal80p and interact with the RNA polymerase

Glucose

If present will inhibit Gal4p

Gal4p

Activator for all gal genes. Works by binding to UASs on DNA sequence. Depends on repression or activation if it binds directly or binds via Gal80p and ATP and Gal3p

Gal regulatory genes

Gal80p

Gal3p

Gal4p

Galactose present and glucose present (effect on expression of gal gene)

Galactose causes Gal3p and Gal1p to be made and inactivates Gal80p

Gal4p is inactivated by glucose

Result is no expression of gal gene

Galactose present and glucose not present

Galactose causes Gal3p and Gal1p to be made and inactivates Gal80p

Gal4p stays active because glucose is not present to inactivate it

Result is the expression of all gal coding genes

Response elements (two types)

Bound by inducible transcription factors that respond to signals

HSE: heat shock response element. Binds a heat shock response promoter to drive transcription

GRE: glucocorticoid response element - enhancer recognized by steroid receptor

Transcription activation domains (3 types)

The domains bind to the mediator complex

Acidic domains

49aa domain with 11 acidic amino acids

Glutamine rich domains

More than 25% glutamine

Proline rich domains

84aa domain 19 of which are prolines

DNA binding domains classes (4 types)

Basically just a receptor that is on an activator that can bind certain things. After binding to whatever it is that it binds to, the activator can then bind to the DNA and promote gene expression

Zinc fingers

Helix turn helix

Helix loop helix

Leucine zippers

Activators general information

They bind to upstream activating sequences

They interact with RNA pol 2 through the mediator

Gene inactivation by histone deacetylase (steps)

Starting from active gene transcription

Repressors displace the activators

Corepressor binds to the RNA polymerase

Histone deacetylase binds to the corepressor and RNA polymerase dissociates with the DNA

Histone deacetylase removes acetyl groups from histones and causes the DNA to be recondensed and inaccessible

Result is deactivation of gene expression

Gene activation by activators and coactivators

Activators bind to UAS's or enhancers

Activators recruit histone modifying enzymes or chromatin remodeling complexes

Activators recruit TFIID to stabilize binding of RNAP 2 and general TFs

Result is gene expression

CpG island methylation

common place of methylation. Cytosine is the one in the DNA that is methylated

Location of methylation at the genome level

Normally methylated at promoters

Methylated DNA

Not expressed as much because RNA polymerase can't bind

How to observe methylated DNA

Use two restriction enzymes. Both will cut at CCGG or CpG islands but only one of them will cut at non methylated sites. Run both for the same piece of DNA. The one cutting for both sites will show all CCGG sites. Compare with the one only cutting non methylated CCGG islands and the ones that show up on there are the non methylated ones then match those up with the other strand and you can find the methylated areas

Methylated genes

Inactive genes and won't be transcribed

Non-methylated/undermethylated genes

Will be expressed

Hypermethylation

Excess of methylation of genes results in the underexpression of those genes and if the gene that is being affected is a tumor suppressing gene then it can result in cancer

Maintenance methyltransferase

Only methylates CG sequences that are base paired with the parental CG bases that are methylated. It will then methylate the CG sequence on the daughter strand. Basically it just add methyl groups to the daughter cells. Result is copying the methylation so the newly made DNA strand is also suppressing certain genes

Features of CpG islands that would favor transcription

Reduced histone H1

Extensive histone acetylation

DNase 1 hypersensitive sites