Lecture 18 - Eukaryotic Transcriptional Initiation

Question 1

Which RNA polymerase are we mostly interested in? Why?

Answer 1

RNA polymerase 2

It is responsible for the transcription of all protein-coding genes.

Question 2

What does RNA polymerase do while reading the DNA template?

Answer 2

RNA polymerase denatures the template to access and read the single strand of DNA in a 3’ to 5’ direction. Doing so, it takes ribonucleotides that are coming into the active site and forms an RNA polymer, which it send out the exit channel in a 5’ to 3’ manner.

Question 3

Which RNA polymerase is insensitive to α-amanitin? Which RNA polymerase is most sensitive to α-amanitin?

Answer 3

RNA Pol I

RNA Pol II

Question 4

What is a distinct component of RNA pol II?

Answer 4

CTD

Question 5

What are the CTD repeats made of?

Answer 5

Each repeat is a stretch of 7 amino acids.

Question 6

What is the difference between phosphorylated and unphosphorylated forms of the CTD?

Answer 6

The phosphorylated form is associated with an elongating/engaged RNA pol II complex while the unphosphorylated form is associated to an inactive RNA polymerase that might be on a promoter region, just hanging out.

Question 7

Approximatelty how many cell types can our cells differentiate into?

Answer 7

200

Question 8

How do we distinguish between different cell types?

Answer 8

We distinguish between different cell types through changes in gene expression.

Question 9

Which stage of transcription is most important for regulating gene transcription? What does this mean?

Answer 9

Initiation is the msot important means of regulating gene transcription.

This means initiation is somehow getting RNA pol II down to promoters of genes in a stage-specific or spatially controlled manner.

Question 10

Which conserved sequence seems to be slightly upstream of the transcriptional start site?

Answer 10

TATA Box

Question 11

What is the TATA box?

Answer 11

The TATA box is a sequence that tells RNA polymerase that the downstream gene it’s supposed to transcribe is coming up.

Question 12

What is the sequence of the TATA box?

Answer 12

The TATA box is quite rich in TATA sequences (TATAAA quite often) but not exclusively, as some variations are present.

Question 13

Where is the TATA box usually found?

Answer 13

The TATA box is usually found 35 base pairs upstream of the transcriptional start site.

Question 14

What single protein is very important for binding to the TATA box?

Answer 14

TATA-box Binding Protein (TBP)

Question 15

How does TBP interact with the DNA sequence?

Answer 15

The saddle-like TBP proteins interacts with the TATA-box and wedges itself into the minor groove of the DNA helix (the conserved C-terminal of TBP binds to the minor groove to distort the double helix), giving rise to a wicked bend in that region of the DNA. This TBP - and a family of other proteins - wedges itself in and interacts with the minor groove, giving rise to a conformational change around the proximal promoter region.

Question 16

Which groove do DNA binding proteins/transcription factors typically interact with? What is an exception to this?

Answer 16

Major Groove

TBP

Question 17

It turns out that most genes actually don’t have a TATA-box. What do they have instead?

Answer 17

“TATA-less” Promoters

Question 18

What protein is needed for class two transcriptions (transcriptions dependent on RNA pol II) with “TATA-less” promoters?

Answer 18

TBP

Question 19

What effect does TBP have on class one (RNA pol I dependent) and class three (RNA pol III dependent) transcription?

Answer 19

TBP makes class one and three proteins more effective.

Question 20

What are TBP associated factors (TAFs)?

Answer 20

They are part of transcription factor II D (TFIID) (the large multi-unit subunit that TBP is a part of) that interact with TBP to contribute to transcription.

Question 21

How were the critical general transcription factors required for all class two transcriptions (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH) found?

Answer 21

They were found using a protein purification system comprising of liquid chromatographic steps (gel filtration, phosphocellulose chromatography, ion exchange chromatography, etc.) in order to separate proteins of siginficant biological interest

Question 22

How do all the class II transcription factors come together at the start of initiation?

Answer 22

TFIID interacts through the TBP core protein with the TATA-box. Once the TBP in its TFIID disguise interacts with the TATA-box, it’s likely that the bent structure is recognized by TFIIB, and the whole complex is subsequenty stabilized by TFIIA. Once these proteins come together to form a quaternary complex, RNA pol II (which is more or less always associated with TFIIF) will recognized the complex (through an interaction with TFIIB) and join on the DNA template. This complex is recognized by TFIIE, which laters acts as a recruitment scaffold for TFIIH, the last factor to come into the complex.

Question 23

What does TFIID do at the start of initiation?

Answer 23

TFIID interacts through the TBP core protein with the TATA box.

Question 24

What does TFIIB do at the start of initiation?

Answer 24

TFIIB recognizes the bent structure of the DNA.

Question 25

What does TFIIA do at the start of initiation?

Answer 25

TFIIA stabilizes the bent DNA structure with TFIID and TFIIB

Question 26

Which class two transcription factor is more or less always associated with RNA pol II?

Answer 26

TFIIF

Question 27

What does RNA pol II interact with to recognize the class two transcription factor complex?

Answer 27

TFIIB

Question 28

What acts as a scaffold for TFIIH?

Answer 28

TFIIE

Question 29

What happens when you add ATP to the class two transcription factor complex?

Answer 29

When ATP is added, the DNA will “melt” and separate into its two single strands.

Question 30

What must be given to the phosphorylated class two transcription factor complex for it to proceed from initiation to elongation?

Answer 30

rNTPs

Question 31

When does the CTD become highly phosphorylated?

Answer 31

It becomes highly phosphorylated when it proceeds from initiation to elongation (due to the addition of rNTPs).

Question 32

What is the denaturing of DNA to form the transcription bubble dependent on?

Answer 32

The denaturing of the DNA to form the transcription bubble is an ATP-dependent step that probably has nothing to do with polymerase directly, but that is dependent on TFIIH.

Question 33

What subunits are found within TFIIH? What are their functions?

Answer 33

XPB - DNA helicase capable of tracking the DNA template in a 3’ to 5’ direction

XPD - DNA helicase capable of tracking in a 5’ to 3’ direction

CDK7 - Cyclin-dependent kinase responsible for phosphorylating the CTD

CyclinH - Cofactor of CDK7

Question 34

Why do XPB and XPD have “XP-“ as part of their names?

Answer 34

XPB and XPD are mutated in patients with classes of a disease called xeroderma pigmentosum (XP), which is why “XP-“ is part of both of their names.

Question 35

What role does TFIIH have outside of initiation?

Answer 35

TFIIH also functions as a DNA repair transcription factor through nucleotide-excision repair.

Question 36

What is the only stage two transcription factor that has ATP-dependent enzymatic activity? What are these activities?

Answer 36

TFIIH

Both the DNA helicases in TFIIH and the phosphorylation of the CTP by CDK7 are ATP-dependent.

Question 37

What does TFIIH do during the transition from initation to elongation?

Answer 37

CDK7 in TFIIH phosphorylates all the serine residues in the repeats of the CTD.

Question 38

What can’t people with xeroderma pigmentosum do? Explain the cause of this?

Answer 38

People with XP can’t go outside in the sun because their cells can’t correct the thymine dimers that will arise due to UV radiation. This is because XPB and XPD have roles in nucleotide-excision repair (NER) and transcription.

Question 39

Why are genes that are heavily transcribed repaired much more efficiently than regions of the genome that aren’t transcribed?

Answer 39

Since TFIIH is reponsible for all class two transcriptions and also responsible for NER, genes that are transcribed more often will encounter TFIIH more often which can efficiently repair anything needed.

Question 40

What happens to TFIIH after the transition from initiation to elongation?

Answer 40

It appears as though TFIIH doens’t go along with RNA polymerase once it transition from initation to elongation.

Question 41

Which transcription factor is most likely involved in transcription-coupled repair?

Answer 41

TFIIH

Question 42

What are the 5 main parts of initiation (in detail)?

Answer 42

1. Eukaryotic promoters contain proximal elements like TATA-boxes that recruit a bunch of general transcription factors largely mediated by the ability of TBP and its associated TAFs to recognize that structure and sit down on that proximal promoter (they bind to the promoter sequence).
2. Once that complex is formed, the DNA goes through a configuration change that leads to the recruitment of a series of other important general transcription factors (TFIIA, TFIIB, RNA pol II with TFIIF, addition of TFIIE, and finally TFIIH). Only when this group of general transcription factors is formed on the promoter, the stable preinitation complex (PIC), can we actually initate transcription.
3. To initiate transcription (in vitro), we give the stable PIC some ATP so that it will open up the promoter region. Denaturing the DNA makes one of these two strands accessible to RNA polymerase and if the NTPs are present…
4. RNA pol II will switch from an initiation phase to an elongating phase.
5. The CTD is phosphorylated.