L11: Transcription initiation in eukaryotes

eukaryotic genes

monocistronic

eukaryotic transcription

• mRNA undergoes additional processing 

• mRNA gets modified to produce mature mRNA

• nuclear export

nuclear export

• export mature mRNA from nucleus to cytoplasm before translation

• interconnected with mRNA modifications

• only properly matured mRNA can be transported

exons

parts of protein CDS that code for protein

intron

• nucleotide sequences that do not code for proteins

• must be removed before translation

3 modifications to become mature mRNA

• addition of 5’ cap

• splicing

• 3’ poly adenylation

bacteria co-transcription

• translation begins while transcription is still happening

• bacterial ribosome attaches to mRNA as soon as start codon becomes available

• direction of transcription and translation is the same

• 5’ to 3’

eukaryotic transcription general mechanism

• same as bacteria

• makes more proteins than bacteria

• more modification steps

how many RNA polymerases do eukaryotes use

3

rRNA

structural components of the ribosome

RNA polymerase I and III

• synthesize functional RNAs

• rRNAs, tRNA

tRNA

pairs aa to triplet codons during translation

RNA polymerase II

• responsible for transcribing all protein-coding genes

• plus other functional RNAs such as snRNA

snRNA

structural components of the spliceosome

what is RNA poly II made up of

12 subunits

largest subunits of RNA poly II

• RPB1

• RPB2

RPB1

• homologous to bacterial beta subunit

• has a long tail like domain at C terminus

RPB2

homologous to bacterial beta prime subunit

what helps RNA poly II bind to its promoter

6 transcription factors

bacteria sigma factor in RNA poly II

no direct homologue but has equivalent things

class II promoter

RNA poly II binds to this

class II promoter core

has 6 promoter elements

draw class II promoter core

x

where is the +1 site in the class II promoter core

in the middle of the Inr sequence

TATA box

• promoter sequence in the class II promoter core

• at the -25 position 

TATA box consensus sequence

TATAAT

BREu and BREd

• B recognition element

• upstream and downstream of the TAT box

Inr

initiator

MTE

motif ten element

DPE

downstream core promoter element

class II promoter core elements

• BREu

• TATA

• BREd

• Inr

• MTE

• DPE

general transcription factors

• GTF

• used to initiate from all genes that use the class II promoter 

• do not regulate a specific group of genes 

TFIID

• binds to Inr, MTE and DPE

• multi subunit protein

• contains over 10 subunits

TBP associated factors

• TAFs

• subunits of TFIID

• TFIID binds using some of its TAFs

TATA binding protein

• TBP

• subunit of TFIID

• binds to TATA box

what happens when TFIID binds to DNA

• TFIIA replaces TAF1 subunit of TFIID

• enables TATA binding protein to bind to TATA box

what happens when TBP binds to TATA box

• bends DNA by a lot

• does not create rep bubble

• flags the location to attact more GTFs

what happens to TBP when it bends DNA

• folds into a symmetrical shape with beta sheets facing the dsDNA major groove

• beta sheet folds into a saddle shape - this bends the DNA

through what does TFIIB bind to the bent DNA

BREu and BREd

TFIIB structure

• long, hook like domain that protrudes out of the structure

• known as B ribbon

what does TFIIF do

• binds to RNA poly II while other GTFs are assembling at the promoter

• also has a hook like domain

• hooks itself onto the TFIIB B ribbon

• secures RNA poly II to the promoter

what does TFIIE do

binds to the promoter to clamp the dsDNA securely onto the protein complex

what does the TFIIH do

• binds the complex

• concludes the assembly of GTFs and RNA poly II onto the class II promoter 

• does not load/stabilize RNA poly II onto the promoter

TFIIH

multi activity enzyme that regulates RNA poly II to enter transcription elongation

write out the steps of RNA poly II binding to the class II promoter

x

6 general transcription factors

• TFIID

• TFIIB

• TFIIA

• TFIIF

• TFIIE

• TFIIH
  

mediator

• huge multi protein complex that regulate entry into elongation

• binds to GTFs and RNA poly II forming the pre-initiation complex (PIC)

what does the mediator do

• facilitate communication between RNA poly and other transcription activators to determine the correct timing to begin elongation

• kepps track of the types of activators that its interacting with

• activates TFIIH

enhancers

• enhancer elements exist upstream of class II promoter

• binding sites for transcription activators

• activators bind to corresponding enhancers

what happens when activators binds to enhancers

• DNA loops over to contact the mediator

• proper activator-mediator interaction starts elongation

how do cells control activators

• cellular conditions

• environmental factors

TFIIH enzymes

• helicase

• kinase

helicase

opens up the dsDNA to generate rep bubble

kinase

• phosphorylates the RNA poly II C terminal domain 

• makes the mediator detach from RNA poly II allowing it to enter elongation

CTD aa sequence

YSPTSPS

how many repeats does human CTD have

52

why can’t CTD fold into secondary structures

• has too many prolines

• all other side chains have an OH group for phosphorylation

what exactly is phosphorylated in CTD

• the 2nd serine in each sequence

• promotes the beginning of elongation

what happens after dna enters elongation

RNA poly II detaches from most of the mediators and GTFs

what helps RNA poly II during elongation

• hundreds of proteins

• these proteins perform various functions related to elongation

• ex. pre-mRNA modification