Lecture 4 - RNA Polymerase, Transcription And Regulation Of Initiation

Question 1

What are the 3 RNA polymerases?

Answer 1

Polymerase 1, polymerase II and polymerase III

Question 2

What do polymerases do?

Answer 2

They transcribe

Question 3

What are the core 5 subunits and are these conserved?

Answer 3

Beta’, beta, alpha 1, alpha, and w

Question 4

What are the steps of transcribing DNA?

Answer 4

Recognise the start and get into position
Start moving
Know when to stop

Question 5

The TFII is found in the pol II promoter. What does it do?

Answer 5

Transcribe factor for pol II

Question 6

What does TFIIB do?

Answer 6

It’s needed at nearly all Promotors of Pol II

Question 7

What does TFIIF do?

Answer 7

Help to position RNA poly II at promoter

Question 8

What does TFIIE do?

Answer 8

Aid in pulling apart two strands of DNA

Question 9

What does TFIIH do?

Answer 9

Help to release RBA pol II to start elongation

Question 10

What is the TBT?

Answer 10

TATA binding protein

Question 11

What does the TBP do?

Answer 11

Bind the TATA sequence in minor grooves of DNA causing a kink in the DNA. This is the major sequence specific protein-DNA interaction during initiation

Question 12

What is the sequence of transcriptional initiation?

Answer 12

TFIID (TBP) binds TATA sequence
TFIIB - recognises BRE element which helps with positioning
TFIIF - stabilises interaction between polII and other factors (TBP) - helps attract TFIIE and TFIIH
TFIIE - attracts and regulates TFIIH
TFIIH - unwinds DBA m, phosphorylase’s Pol II tails (DNA helipads and ATPase activity, kinase activity)

Question 13

After initiation how does elongation occur after DNA unwinds?

Answer 13

RNA pol II synthesises short lengths of RNA

Period of abortive initiation

Shifts ti elongation when phosphate group are added ti tail of RNA poly II - CTD

Disengages from GTFs

Acquires more proteins belong transcribe

Most of GTFs are released available to initiate another round of transcription

Question 14

Activators - what does the basal apparatus do?

Answer 14

Determines the start point for transcription

Question 15

What do activators determine?

Answer 15

The frequency of initiation/strength of promotor

Question 16

What do activators have with DNA

Answer 16

Binding activity

Question 17

What do activators contact?

Answer 17

Other basal transcription machinery or commercial-activators

Question 18

How do some components of the transcriptional apparatus work?

Answer 18

By changing chromatin structure

Question 19

What are examples of changing chromatic structure?

Answer 19

Covalent history modification
Nucleosome remodelling
Nucleosome removal
Histone replacement

Question 20

There is different modes of Repression - what is the competitive DNA binding mode of action?

Answer 20

Activator binds near the repressor stopping the bringing which triggers the activator

Question 21

There is different modes of Repression - how does masking the activation surface work?

Answer 21

Blocks the activator to bring and roaming to factors

Question 22

There is different modes of Repression - how does direct interaction with the general transcription factors work?

Answer 22

This could directly bind to factors

Question 23

There is different modes of Repression - How could the DNA become less accessible in order to stop the activation?

Answer 23

Remodelling the Nucleosome so the promotor cannot be reached this is fine through recruiting histones deacetylase or Histone methyl transferase.

Question 24

How do activators open up chromatin to make the dna more accessible?

Answer 24

Bind to specific elements of promotors and recruit Histone acetylase which lets transcription factors bind to promotors

Question 25

What do mediators do?

Answer 25

They mediate transcriptional activators and Pol II

Question 26

What does the mediator complex do?

Answer 26

Bind pol II and it’s initiation factors and can enhance recruitment of pol II ti activator bound DNA in vivo

Question 27

What are the infection triggers for type 1 interferons?

Answer 27

NFkB
IRF-3/7
Jun/ATF

Question 28

What do the infection triggers of type 1 interferons do?

Answer 28

Activate IFN-B which is secreted by infected cells to warn/program y infected cells in an antiviral state

Question 29

Where do activators bind?

Answer 29

Upstream of the TATA box in a place called the enhancesome

Question 30

What method do you use to detect in vitro regions of DBA bound to protein?

Answer 30

Foot printing/ EMSA

Question 31

What method do you use to identify regulatory proteins bound in vivo?

Answer 31

ChIP

Question 32

How do you do DNA foot printing?

Answer 32

Synthesise or amplify DBA if interest

Label

Incubate with a protein

Use DNase or hydroxyl radical to cleave it

Visualise the resulting pattern along side a sequencing ladder

Question 33

How do we identify where DNA binding proteins bind?

Answer 33

DNA binding changes mobility of dna on gel

You need purified protein and an antibody

Question 34

What does ChIP determine?

Answer 34

Determine all regulatory sequences occupied by a given transcription regulator

Can be used to determine positions along genome bound by modified histones

Question 35

How do you do a ChIP?

Answer 35

Get a protein in vivo and cross link protein to the DNA so it’s covalent attached.

Then lyse and break the the DNA. You then precipitate the DNA and use the antibody to get rid of the cords link and see what you brought down

You can do this through a PCR

Question 36

Why add chemicals to DNA?

Answer 36

To get rid of the proteins protecting it