L1- eukaryotic promoters and RNA polymerases and PIC assembly

Gene expression definition

Process by which information in genes (DNA) is decoded into protein

How many steps is gene expression and what are tehy

2: transcription and translation

Start and end product of transcription

DNA → ss RNA (mRNA)

Start and end product of translation

mRNA→ protein

What is the enzyme involved in trancription and what product does it make

RNA polymerase- converts DNA to mRNA

What subunit does RNA polymerase in prokaryotes have adn therefore what is it called

Sigma 70 → with rna polymerase= holoenzyme

Holenzyme

RNA polymerase w/ sigma 70

Stages of transcription in prokaryotes (6) FINSH ONCE RECAP WACTHED

• starts as a closed ocmpelx

• Holenzyme makes contact with DNA in the -35 to -10 region

• Melts DNA over the trasncription start site giving rna polymerase access to template strand

• Open compelx is formed

• Elongation of mRNA strand

• After 5-10 nuecltodes sigma 70 is released and elongation stops

Promoters definition

Cis acting DNA regulatory element through which transcription is initiated and controlled

What does the promoter dictate

Where and how efficiently the RNA Polymerase can bind so therefore influences how much protein is made

Where are prokaryotic promoters usually foudn

-35 and -10 region

Consensus sequences

Idealised bacterial promoters

Example of consensus sequence at -35

TTGACA

example of consensus sequence at -10

TATAAAT

Exampel of start site in prokaryotes

TG/AT

Roughly what number of base pairs are between the -35, -10 and +1 start site region

-35 → -10 = 16-18bp

-10 → +1 = 5-8bp

What can th eukaryotic promoters region be split into

• core/ basal region

• Regulatory region

which region in eukaryotic promoters does rna polymerase bind CHECK L

Core/basal region

Approx where is the tata box located

-30 kb

Where is the initiator element found in euk promtoer

-2 to +4 at start site

Key feature of initiator region

Rich is A and T bases

Core/ basal promoters elements (5)

• tata box

• Initiator (Inr)

• Motif ten element (MTE)

• Downstream core promoter element (DPE)

• TFIIB recognition element (BRE)

Where is the tata box located

-31 - -26

Where is the Inr located

-2 - +4

Where is teh MTE located

+18 - 28

Where is teh DPE located

+28 - +32

Where is the BRE located

-37 - -32

Sequence of the tata box

T-A-TA-A/T-A-A/T-A/G

Sequence of the initiator (Inr)

Y-Y-A^+1-N-A/T-Y-Y-Y

Sequenc of the MTE

C-G/C-A-A/G-C-G/C-G/C-A-A-C

Sequence of DPE

A/G-G-A/T-C-G-T-G

Sequenc of BRE

G/C-G/C-G/A-G-G-C-C

Where are CpG islands found

In eukaryotic promoter regions

What do 60-70% of protein coding genes in mammals lack

Obvious TATA and Inr regions

Rate of transcription in eukaryotes

Low rate and at several start sites

What are CpG islands assocated with

Associated with regions with a high frequnecy of CG sequences

How is dna slightly different in CpG islands

Is methylated

What commonly follows C residues in mammals

Often a G and are methylated ( g to 5-Methyl C )

What do C residues in CpG often escape

Methylation (hypomethylated)

How does methylation affect transcription

Methylation of CgP islands is associated with silencing, hypomethylation is associated switching on

What can the regulatory region be divided in to

The proximal region

What can the regulatory region be divided into

Proximal region and the enhancer region

Where is the proximal region foudn

Often wihtin 1 kb of the core reigon but can alos be >1kb

What feature are there in the regulatory region (2)

• UAS/enahncer

• URS/silencer

UAS

Upstream activator binding site

What does the UAs do

Are activator binding sits so proteins will stimulate transcrition

URS

Upstream repressor binding sites

Are eukaryotic or prokaryotic promoters more complex

Eukaryotic

2 tools for identifying comparison

• sequence comparison

• Reporter analysis

What method IDed the TATA box

Sequence comparison

Feautres of sequence comparison (3)

• look for common patterns

• Frequency of ases in a particualr position are compared

• But tells you nothing about the function o the dna/ protein

Reporter analysis

Study how a a regulator sequence works and the important elements in it

How deos reporter analysis work

Takes sequence and use it to drive expression of reporte genes

Reporter genes

Encode proteins whose levels can be easily measured eg enzymes such as GFP, luciferae, LacZ

How can reporter analysis work

Build a plasmid wiht promoter of interest driving expression of a reporter gene, trasnfect it into cells, regulatory sequence drives transcription, gene is express, can use enzymatic acivity to easily measure amount of protien to report on activity of regulatory sequence/ gene expression

When can reported be used to identify (4)

• when a gene is expressed

• Where it is expressed

• What signals ti responds to

• What factors and sequence control its expression.

Where is rna pol I found

Nucleolus

Where is rna pol II found

Nucleus

Where is rna pol III foudn

Nucleus

Target genes of rna pol I

rRNA (28S, 18S, 5.8S)

What are the target genes of RNA pol II

mRNA, snRNA, miRNA

What are the target genes of RNA pol III

tRNA, 5S RNAs, U6 RNA, 7S RNA

What does bacterial RNA polymerase require to recognise promoter DNA in bacteria

Sigma factor

What do eukaryotes have instead of sigma factor (6)

• TFIIA

• TFIIB

• TFID

• TFIE

• TFIIF

• TFIIH

• = general transcription factors

What do genearl trancription factors do (5)

• rna pol specific

• Multi component factors

• Form a complex on the core promoter

• Recruit rna pol II

• Direct initiation at start site

Features of general transcrition facotrs (5)

• rna pol specific

• Multi component facotrs

• Forma a complex on the core promoter

• Recruit rna pol II

• Direct intiation at start site

• Majoring are multiple polypeptides

PIC

Pre initiation complex

PIC assembly steps (5) CHECK

• TFIID binds to TATA box

• Brings in TFIIA and TFIIB

• TFIIB makes direct contact with rna polymerase and recruits it to the promoter

• TFIIE and TFIIH bind

• TFIIH is critical complemetn of PIC formation

What opens the dna at the start site

Helicase activtiy of TFIIH

CHECK

What does helicase require

ATP hydrolysis

What happens once helicase has separated template strand at start site

DNA pol 2 begins transcribing → get formation of first phosphodiester bonds in rna trasncript and is extensively phosphorylated on the c terminal domain

What si teh c terminal domain

A series of repeats located at hte c terminal end of the largest (beta homologous subunit) of pol 2

What happens to certain TF during trasnciption invitation as polymerase moves down template (3)

• TFIID and TFIIA may stay behind

• TFIIB, TFIIE and TFIIH are released

• TFIIF moves down the template with pol II

Function and number of subunits of TFIID

• 13

• Binds to TATA box in core promtoer and recruits TFIIB

Function and number of subunits of TFIIA

• 3

• Stabilises TFIID binding and has anti repression fucntion

Function and number of subunits of TFIIB

• 1

• Recruits rna pol II- TIIF and sitmualtes RNA pol II elongation

Function and number of subunits of TFIIF

• 2

• Assists TFIIB recruit RNA pol II and stimualtes RNA pol II elongation

Function and number of subunits of TFIIE-

• 2

• Helps recruit TFIIH and modulates TFIIH activity

Function and number of subunits of TFIIH

• 9

• Promtoer melting and clearnace

• CTD kinase acvitity

• DNA repair coupling

Which 2 GTFs intitiate txn

TFIID and TFIIH

Features of TFIIH (3)

• composed of 9-10 subunits

• Can be divided into CORE and CAK

• Contains ATPase XPB (SSL2) that plays a key role in promtoter melting

What does the CAK module contain in TFIIH

Contains one of the kinases that phosphorylase’s the CTD of rna pol 2

How does SSL2 (the atpase) work

Uses the energy from atp hydrolysis to push dna into the cleft where ran polymerisation is catalysed, causing torsional stress that contributes to promoter melting and open complex formation

What sit eh central rna pol2 transcrition factor

TFIID

Wwhat si TFIID in simple terms

Tata binding protein + tbp assocaited factors (TAFs)

What is tbp

A central subunit of TFIID that froms complex with other assocaited factors (TAFs)

How can tbp be described and why

As a molecular saddle → sits above dna making direct contact with the TATA box → dna binds to under side of tbp

What can tbp do tha TFIID cannot

Can direct assembly of thePIC on a tata containing promoter → TAFs not required

Whtat can TBPs not do (2)

• cannot alone direct pic assembly on a tata less promoter

• Cannot alos support ‘activated’ transcription

What do TAFs do (2)

• promoter interaction of TFIID with basal promoter elements eg taf1 can interact with Inr, taf6/9 cann interact with DPE

• Interact with activators to promote transcription initiation

How do TAFs work

Form ethe need strucutres to interact with other elements