Chromatin Modifications Flashcards
What is euchromatin?
In interphase the chromosomes are spread out in the nucleus and the spread out parts are euchromatin
What is heterochromatin?
Some parts of chromosomes are condensed in interphase (e.g: centromeres) :heterochromatin
DNA is heterochromatin inaccessible in…
Transcription
What is X-chromosome inactivation?
In female mammals, only one X chromosome is active; the other is always condensed and inactive
What is different about the structure of expressed genes? How is this shown? How are they packaged?
They have a more open structure than others, shown by greater sensitive to DNase. In this state, the only packaging is into nucleosomes
The beta-globin gene is an example of an expressed gene. Example how
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
What are the two main processes of the opening up of chromatin?
. Histone modification
. Chromatin remodelling complexes
Explain histone modification (opens up chromatin)
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
Explain chromatin remodelling complexes
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
Explain the essential concepts of chromosomes and chromatin
. 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
What are the three types of RNA polymerase in eukaryotes and what is there function?
. 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)
What are the similarities between RNA polymerase in bacteria and eukaryotes?
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
Describe the transcription complex
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
What are the specific initiation requires of the transcription complex?
. 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
What is the transcription complex?
Key sequence for initiation of transcription in promoter of many genes: TATA box (consensus TATAAA; normally 8bp all A/T)
What does the TATA box do?
Directs RNA polymerase to the correct starting point
Where is the TATA box found?
About 25bp upstream of the transcription start point
How is the start site to initiate transcription identified? Explain how it is initiated
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
What is TF11D?
Contains about 8 polypeptide subunits, sometimes called TAFs, for TATA box associated factors
For the many genes have do not have a TATA box how do you know where to begin transcription?
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
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?
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
What is the typical sequence of steps in transcription initiation ?
- Preparation for transcription
- Assembly of the transcription complex
- Release of RNA polymerase
Describe the preparation for transcription (opening up the chromatin and making it accessible for transcription)
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
Explain the assembly of the transcription complex
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
Explain how RNA polymerase is released at the end of transcription
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
Summarise eukaryotic transcription initiation
. 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
