4L6&7 Eukaryotic Transcription

eukaryotic gene organization

each gene has a promoter(s) and terminator(s)

exon = included, intron = excluded in primary transcript

overall expression of functional protein is regulated at

• transcription

• rna processing

• mRNA turnover

• translation

• posttranslational modification

• cellular trafficking

• protein turnover

chromatin

eukaryotic chromosomal material of DNA, proteins (histones), and RNA

amorphous in G0 and interphase,

chromatin remodeling

heterochromatin v euchromatin

hetero = more condensed, inactive, 10% chromatin, often in structures, dark staining

eu = normal, transcription, light staining

nucleosome

basic unit of chromatin contains 8 histones to make up histone core

in euchromatin, nucleosomes look like beads on a string

10nm fiber

nucleosome beads on a string (10nm is diameter of nucleosome)

wraps around histone twice

histones

positively charged,

DNA-histone core contacts are sequence independent

form electrostatic interactions and H bonds with negative backbone

histone core

eight histone molecules, 2 copies of H2A, H2B, H3, H4

one core per nucleosome

histone H1

locks DNA to nucleosome

tails of histones

play key role in forming contacts between nucleosomes,

most of histone modifications here

epigenetics

chromatin structure is a product of epigenetic marks (histone mods and DNA methylation) and trans-acting transcription factors

histone tails

histone tail has H3 and H4 in N termini,

N and C termini of H2A H2B,

post translationally modified

histone tail modifications

acetylation = in areas of open chromatin, HAT acetylates lysine

hypermethylation = often associated with closed chromatin, HMT methylates lys arg

HAT

histone acetyltransferase, acetylates lysine in histone tails

HDAC

histone deacetylase removes acetyl groups from histone tails

HMT

histone methyltransferase methylates lysine and arginine in histone tails

histone acetylation

neutralizes positive charge on lysine in histone tails

reduces electrostatic nucleosome-DNA attraction

helps recruit chromatin remodeling complex

helps transcription activation

histone methylation

methylation retains positive charge on lysine/arginine

can activate or silence, typically closed chromatin

bind sites for enzyme

transcribed genes are

more diverse

histone code

trimethyl lysine 27 on histone 3 = H3 K 27 me3

DNA transcription regulated by posttranslational modifications to histones

to designate a modification, (histone protein) (amino acid letter) (# position of aa at N terminus) (covalent modification)

DNMT

DNA methyltransferase

CpG island

hypermethylation can silence promoter

cluster of CG bonds that can be methylated

methyl group of 5methyl cytosine protrudes into major groove

affects binding transcription

DNA methylation

NOT histone methylation,

CG, C can be methylated > CpG

euk transcription requires positive regulation

default is off, must be activated

activators, architectural regulators, chromatin mod, remodeling proteins, coactivators, general transcription factors

architectural regulators

help DNA looping, bind between promoter and activator/repressor

abundant in chromatin

HMG = high mobility group, help remodeling and activation

high mobility group

help chromatin remodeling and transcription activation

coactivators

help communication between activators, Pol2, and general transcription factors

repressors may bind in their place

transcription start site +1

has promoter proximal elements upstream -40>-200

enhancers promoter distal positions

DNA binding domain is made to position a ____ into the ____

recognition helix; major groove

most eukaryotic sequence-specific DNA binding proteins have one of these motifs:

helix-turn-helix, homeodomain

classical zinc finger, nuclear receptor zinc finger,

leucine zipper proteins, helix-loop-helix proteins

**all have recognition alpha helix

DNA binding domain

has TF bind site, activation domain, made up by TFs

zinc finger

30 aa long, elongated loop held by Zn2+ ion;

antiparallel B sheets, 2 cysteines, turn, alpha helix, 2 histidines

cysteines and histidines coordinate Zn to make 3D structure

prokaryotic v eukaryotic transcription factors structure

we have monomer with multiple DNA binding sites,

prokaryotes have to have dimers

the more recognition helixes,

the more specific

leucine zipper

2 roles, bind DNA specifically, and protein-protein interactions to bring subunits together;

zipper region has lots of zipper, interacts with Hphobic on other helix

combinatorial control

mixing and matching protein family variants

activation domain

interact with other proteins to recruit them to gene and build the complex for transcription;

coactivators, HAT, CRC (chromatin remodeling complex), mediator, PIC (preinitiation complex);

area of protein-protein interaction

chromatin remodeling

HATs do covalent modifications, acetylate histone tails

CRC use energy to push nucleosomes

SWI/SNF

a CRC that binds to nucleosomes, removes histone proteins, opens to recruit other proteins

pioneering transcription factor

can access DNA as heterochromatin (only one, very rare);

opens it through activation domain, recruits HATs and CRCs

eukaryotic transcription initiation

1. activators (TFs) bind to regulatory sites

2. activation domain recruits coactivators: HATs, CRCs, TFs

3. recruits mediator, bridges activators and RNA polymerase at promoter

4. recruit RNA poly to recruiter

promoter proximal sites

near promoter

mediator

acts as bridge between activators and RNA pol 2 at promoter;

large complex, major eukaryotic coactivator (doesnt bind to DNA)

binds different activators with 30 polypeptides, binds to CTD

GR nuclear hormone receptor example

1. GR binds to HRE at promoter proximal sites or enhancers

2. GR activator domain recruits coactivator with HAT activity

3. recruits mediator (MED1), which interacts with PIC at promoter.

eukaryotic RNA polymerase

RNA poly 2, makes mRNA and ncRNA (c term tail)

RNA poly 1 makes rRNA

RNA poly 3 makes tRNA and ncRNAs

similar to prokaryotic core enzyme

core enzyme v holoenzyme in prokaryotes

holoenzyme has sigma, which recognizes promoter sequences

why do we have to bring RNA polymerases to the promoter in eukaryotes?

it can’t find the promoter by itself

general TFs

help gets pol 2 to the promoter

TFII___

TFs

proteins that help pol 2 form active transcription complex

TF2D

read and binds to promoters

made of TATABP and 13 TAFs

TATABP

locates and binds TATA

TAFs = TBP-associated factors,

transcription initiation & locating promoters

1. TF2D finds it

2. recruits TF2A maybe

3. recruits TF2B which binds to DNA and TBP,

4. TF2F & RNA pol 2 binds TF2B

5. TF2E and TF2H enter

6. mediator gets everything together

7. TF2H Plates cystines of CTD of pol 2

8. elongation, TF2E and TF2H leave

9. leaves promoter, transcribes until terminator

TF2F

binds RNA pol2 to TF2B

TF2H

has helicase (needs ATP) and kinase;

Plates pol 2 at CTD

when does transcription start for real

mediator bridges activators and pol 2

example of gene activation in yeast

starts as heterochromatin, is activated to beads on a string

negative gene regulation

competitive binding, corepressor, altering assembly of PIC, provide dock site for HDAC

corepressor

prevents interaction with mediator

HDAC

condenses chromatin,

HDAC deacetylates lysine, restoring positive charge

easily reversed

heterochromatin

HMTs (histone methyl transferase) help condense with chromatin associated proteins

HP1 = docking site for HMTs