Chromatin Modifications

Question 1

What is euchromatin?

Answer 1

In interphase the chromosomes are spread out in the nucleus and the spread out parts are euchromatin

Question 2

What is heterochromatin?

Answer 2

Some parts of chromosomes are condensed in interphase (e.g: centromeres) :heterochromatin

Question 3

DNA is heterochromatin inaccessible in…

Answer 3

Transcription

Question 4

What is X-chromosome inactivation?

Answer 4

In female mammals, only one X chromosome is active; the other is always condensed and inactive

Question 5

What is different about the structure of expressed genes? How is this shown? How are they packaged?

Answer 5

They have a more open structure than others, shown by greater sensitive to DNase. In this state, the only packaging is into nucleosomes

Question 6

The beta-globin gene is an example of an expressed gene. Example how

Answer 6

Globin genes are transcribed only in erythronium cells, the precursors of red blood cells
. Liver: globin genes giggly packaged
. Erythronium cells: globin genes sensitive to DNase

Question 7

What are the two main processes of the opening up of chromatin?

Answer 7

. Histone modification

. Chromatin remodelling complexes

Question 8

Explain histone modification (opens up chromatin)

Answer 8

Especially histone acetylation by histone acetylases: addition of acetyl groups (CH3CO) to lysines residues. Histones can be reversibly cetylated on some of their lysines. Acetylation removes a positive charge from the histone. Less positive charge makes the interaction looser, loosening nucleosomes because of reduced electrostatic attraction

Question 9

Explain chromatin remodelling complexes

Answer 9

Increase accessibility of DNA. Active process, requiring energy from ATP hydrolysis. To allow access by other DNA binding proteins (DBP), nucleosomes can be partly unwrapped, moves or removed completely. So, repositioning of the nucleosomes and again loosening of the interaction between the DNA and the nucleosome

Question 10

Explain the essential concepts of chromosomes and chromatin

Answer 10

. Eukaryotic DNA is organised into linear chromosomes
. There are multiple levels of packaging of DNA, starting with nucleosomes
. For transcription to occur, DNA must be made accessible by histone modification and chromatin remodelling

Question 11

What are the three types of RNA polymerase in eukaryotes and what is there function?

Answer 11

. RNA polymerase 1
- transcribes ribosomal RNA genes
. RNA polymerase 2
- transcribes genes coding for proteins
. RNA polymerase 3
- transcribes bees coding for small RNA such as tRNAs
(Each type has a core enzyme containing >10 polypeptides; some are shared among the different types)

Question 12

What are the similarities between RNA polymerase in bacteria and eukaryotes?

Answer 12

All forms of eukaryotic RNA polymerase have subunits that correspond to the alpha, beta, beta prune subunits of bacterial RNA polymerase. The structures of bacterial and eukaryotic RNA polymerase overlap

Question 13

Describe the transcription complex

Answer 13

Purification of eukaryotic RNA polymerase gives core enzymes, which do not initiate transcription specifically. In eukaryotes a transcription complex of at least 50 polypeptides is required for transcription in vivo

Question 14

What are the specific initiation requires of the transcription complex?

Answer 14

. Enzymes that alter chromatin structure
. Specific activator proteins
. Mediator, which links RNA polymerase to activator proteins
. Nasal or general transcription factors that help RNA polymerase bind to promoters and initiate transcription. Basal factors for transcription by RNA polymerase 2: TF11A, TF11B, etc.
. RNA polymerase core enzyme

Question 15

What is the transcription complex?

Answer 15

Key sequence for initiation of transcription in promoter of many genes: TATA box (consensus TATAAA; normally 8bp all A/T)

Question 16

What does the TATA box do?

Answer 16

Directs RNA polymerase to the correct starting point

Question 17

Where is the TATA box found?

Answer 17

About 25bp upstream of the transcription start point

Question 18

How is the start site to initiate transcription identified? Explain how it is initiated

Answer 18

The TATA box binding protein (TBP) binds to the TATA box. Binding puts a wedge into the minor groove of the DNA molecule, twisting the DNA so that it bends at an angle of about 70 degrees. Pushes the DNA apart and opens it up for transcription. TF11D: TBP forms part of a complex called TF11D

Question 19

What is TF11D?

Answer 19

Contains about 8 polypeptide subunits, sometimes called TAFs, for TATA box associated factors

Question 20

For the many genes have do not have a TATA box how do you know where to begin transcription?

Answer 20

Some have one or more other sequences that define where it starts (and can be present in genes that have a TATA box as well). These bind to TF11B or to other subunits of TF11D. Nevertheless, TBP is required for transcription and bends DNA. Any gene will have a full set or a subset of these sequences

Question 21

For genes that do not have a TATA box or any of the other sequences that define where transcription begins. How do these genes work?

Answer 21

They tend to be genes coding for ‘housekeeping’ proteins that are required in all or most cells in relatively small amounts. Transcription of these genes typically does not start at a specific base: dispersed initiation over 50-100bp

Question 22

What is the typical sequence of steps in transcription initiation ?

Answer 22

1. Preparation for transcription
2. Assembly of the transcription complex
3. Release of RNA polymerase

Question 23

Describe the preparation for transcription (opening up the chromatin and making it accessible for transcription)

Answer 23

a. When gene is to be made available for transcription, transcription activator proteins bind to specific sequences of DNA, displacing nucleosomes
b. To increase accessibility, histones are acetylated and chromatin is remodelled
c. When gene is about to be transcribed, additional activator proteins bind to additional specific sequences in its DNA

Question 24

Explain the assembly of the transcription complex

Answer 24

Activator proteins can bind to TAFs and to mediator, which helps RNA polymerase bind to the promoter
Steps, omitting mediator:
a. TF11D (TBP+TAFs) binds to promotor (bind the when needed). Binding of TF11A and TF11B stabilises the complex
b. RNA polymerase 11 (Pol 11) and TF11F bind, forming core pre-initiation complex (PIC)
c. Binding of TF11E and TF11H completes the complex

Question 25

Explain how RNA polymerase is released at the end of transcription

Answer 25

a. DNA is opened forming transcription bubble (involves opening up the DNA- separating the DNA); requires ATP hydrolysis by TF11H. TF11H also phosphorylates Pol 11
b. Transcription starts, but commonly pauses
c. Elongation factors (a set of polypeptides) join with RNA polymerase and initiation factors dissociate, giving elongation complex. Cap is added to the 5’ end to protect it)
Phosphorylation of elongated carboxyl terminal domain (a region of RNA polymerase) of Pol 11 by TF11H and positive transcription elongation factor-b (P-TEFb) is required for real ease of RNA polymerase and the DNA can be transcribed

Question 26

Summarise eukaryotic transcription initiation

Answer 26

. Chromatin must be made accessible
. Three kinds of RNA polymerase, all related to bacterial RNA polymerase
. Transcription factors help RNA Polymerases bind and initiate transcription
. Promoters for RNA polymerase 11 can have different kinds of sequence; in some cases, initiation is dispersed
. RNA polymerase and associated proteins form the transcription complex
. After the transcription complex forms, TF11H and PTEFb phosphorylate RNA polymerase 11, releasing it