Lecture 27. Chromatin and epigenetic effects Flashcards
When are prokaryotic RNAs translated into proteins ?
Co-transcriptionally - as soon as they are made
Why are prokaryotic RNAs translated as soon as they are made ?
Due to the absence of the nuclear envelope
When are eukaryotic RNAs translated ?
They are first modified and then transported from the nucleus to cytoplasm
Why is eukaryotic RNA transported into the cytoplasm for translation?
Eukaryotic DNA is compartmentalized within the nucleus
What needs to happen to eukaryotic RNA to be transported into the cytoplasm ?
Addition of sequences to the 5’ end of the RNA (5’cap) as well as the 3’ end (the polyA tail)
What is the function of adding sequences to the 5’end and the 3’end of eukaryotic RNA ?
- Regulates stability of mRNA molecules
2. Exert a level of control over gene expression that prokaryotes don’t have
What do eukaryotic RNA undergo that prokaryotic RNA do not ?
Splicing
Where does splicing occur ?
In the nucleus
What is the purpose of splicing ?
Remove introns
What is an advantage of splicing to eukaryotes ?
Adds another layer of control and complexity over eukaryotic gene expression
What is the function of eukaryotic mRNA modification ?
- Stabilizes mRNA
2. Facilitate mRNA export from the nucleus
What does mRNA from higher eukaryotes undergo ?
Splicing by small nuclear RNAs
What function does splicing by small nuclear RNAs in mRNA in higher eukaryotes have ?
- Removes introns
2. Generate splice variants of genes
What do eukaryotic RNA polymerase require ?
Transcription factors
What is eukaryotic RNA polymerase’s transcription factors access to DNA regulated by ?
- Histones
2. DNA binding proteins
Why is gene expression in eukaryotes constitutively off at most promoters ?
- The requirement for specific and general transcription factors
- The physical inaccessibility of gene promoters
What do specific and general transcription factors regulate ?
The binding of the RNA polymerase complex to the promoter
What makes gene promoters physically inaccessible ?
Histones
What do histones form ?
Particles called nucleosomes
What do nucleosomes bind tightly to ?
DNA
What is the structure of a nucleosome ?
Has 200 base pairs of DNA wound around it and other DNA binding proteins
How do DNA binding promoters regulate the binding of RNA polymerase to the promoter region ?
Compacts DNA into a closed state that blocks access of the RNA polymerase complex to the promoter
What do eukaryotic RNA polymerases require that prokaryotic polymerases do not to initiate transcription ?
Transcription factors to be bound to the promoter region
What is RNA polymerase 1 responsible for ?
Generating rRNA precursor molecules
What is RNA polymerase 2 responsible for ?
Generating mRNA and RNA
What is RNA polymerase 3 responsible for generating ?
Small RNAs such as tRNA and 5S rRNA
What do RNA polymerase 2 promoters regulate ?
mRNA synthesis
Where are RNA polymerase 2 promoters located ?
-30 to the transcription start site at a TATA box
What is the actual sequence of of the TATA box in RNA polymerase 2 promoters ?
TATAAAA
Do all polymerase 2 promoters have TATA boxes ?
No
Where is the CCAAT box found ?
-75 region
What is the upstream elements of polymerase 2 promoters ?
GGCCCAATCT element or CCAAT box
What is the general rules about promotes recognized by RNA pol II ?
Typically contain a TATA box and at least one other upstream element
What is the purpose of transcription factors for eukaryotic polymerases ?
need transcription factors to assist in eukaryotic RNA polymerases are recognizing the promoter regions of genes on their own
What are the two types of transcription factors ?
- General
2. Gene specific
What are the 7 general transcription factors that collaborate with RNA Pol II ?
- TFIIA
- TFIIB
- TFIID
- TFIIE
- TFIIF
- TFIIH
- TFIIJ
What does TFIID contain ?
Many subunits including the TATA box binding protein
Where does the TATA box binding protein (TBP) bind to ?
The TATA box bringing TFIID
What does the DNA binding domain of the TBP consist of ?
A large B-sheet that forms a saddle shaped structure that sits astride the DNA in the region of the TATA box
What does the binding of TBP to the TATA induce ?
A dramatic distortion
What does TBP binding cause structurally ?
A large kink in the DNA helix causing the DNA duplex to unwind over an 8 base pair span
What does TBP binding allow access of ?
Allows access of the RNA polymerase to the template
What does TFIID serve as ?
A platform for organisation of other transcription factors which then bind in the following order; D, A, B, F+ Pol II, E, H and J
What order to transcription factors bind ?
D, A, B, F + Pol II, E, H, J
What happens upon assembly of the RNA Pol II preinitiation complex ?
The C-terminal domain of the largest subunit of RNA polymerase II becomes heavily phosphorylated
What is most likely the largest subunit of RNA polymerase II that becomes heavily phosphorylated ?
TFIIH
What is TFIIH ?
A kinase
What does the phosphorylation of the largest subunit of RNA polymerase II do ?
A trigger that uncouples the polymerase from transcription factors allowing the polymerase to move along the DNA template
What are general transcription sufficient to enable ?
A basal level of transcription
What are some gene-specific transcription factors ?
- Myc
- Max
- Fos
- Jun
- NfkB
- NFAT
What are the 4 classes that contain motifs that allow interaction between the transcription factors and the major grooves of DNA ?
- Zinc finger motif
- Helix-loop-helix motif
- Leucine zipper motif
- High mobility group box motif
What is the purpose of the four motifs ?
Allow recognition of specific DNA sequences by the transcription factors
What do most motifs contain ?
A segment (often alpha helix) that is inserted into the major groove of DNA
What do motifs that contain a segment recognise and form ?
- Recognise the side chains of the bases lining the groove
2. Forms non-covalent interactions with the side chains
What makes the otherwise weak strength of the contact between the transcription factor and the DNA very high ?
- The number of contacts
2. The overall strength and specificity of the transcription factor DNA interaction
Where are enhancer sequences often found ?
Thousands of base pairs away from a promoter region that the enhancer controls
What is chromatin looping facilitated by ?
Specific transcription factors and mediators (associated proteins)
What is the function of chromatin looping ?
Brings the promoter region in close proximity to the TATA box and the associated RNA pol II complex
What an histones be modified by ?
- Acetylation
- Methylation
- Phosphorylation
What do modifications that neutralize the positive charged on histones do ?
Reduce their affinity for DNA
What is a histone code ?
The number of possible patterns of histone modifications
What is another name for the modification of the histone c-terminal tail
Acetylation
What is the enzyme involved in acetylation ?
Histone acetyltransferases
What is the enzyme responsible for the reverse modification of acetylation ?
Histone deacetyltransferases
What does acetylation lead to ?
A more open chromatin state due to neutralization of the negative charges on histones thereby loosening the histones contact with DNA
What do histones associated with more active genes tend to be rich in ?
Acetyl modifications
What can some histone modifications also recruit ?
Proteins for example polycomb proteins
What is epigenetic control ?
Inherited modifications that are not present in a DNA sequence
What is gene expression regulated by ?
DNA binding proteins called transcriptional regulators as well as the accessibility of the stretch of DNA harbouring the gene (chromatin state)
What are nucleosomes ?
Histone octamers
What causes nucleosomes to be moved off promoters to facilitate the initiation of transcription ?
Switch/Sniff complex or SWI/SNF complex
What do all eukaryotic cells contain and what its function ?
A large multi subunit complex that can disrupt histone-DNA interactions in an ATP dependent manner and allow binding of transcription factors to gene regulatory regions
How was the SWI/SNF complex discovered ?
Screen genes involved in growth on sucrose and screens the genes involved in the ability to switch mating type. One of the genes was found in common
What is the function of SWI/SNF complex ?
Bulldoze histones off promoters and facilitate access of the RNA Pol complex to the promoter
How many proteins is the yeast SWI/SNF complex comprised of ?
11
What does the yeast SWI/SNF complex act as ?
An ATP driven nucleosome plough to facilitate transcription
How do certain transcription factors function ?
By attracting the SWI/SNF complex to the promoter to facilitate transcription
Where are 1% of nucleotides methylated at ?
Cytosine residues
What does the methylated nucleotides produce ?
5-methylcytosine
What does the 5-methylcytosine modification act as ?
A molecular tag that allows certain regions of DNA to be regulated differently than others
Where are almost all methylated cytosines found ?
CG dinucleotides within symmetrical sequences
Where do symmetrical sequences occur (methylated) ?
Tend to be clustered in G rich regions called CpG islands close to gene regulatory regions
Where are unmethylated CpG islands found ?
Near active gene promoters
What is methylation likely involved in ?
Silencing gene expression
What is methylation in vertebrates ?
Dynamic
What does DNA methylation correlate with ?
Gene expression patterns
How does methylation inhibit transcription ?
- By interfering with the recognition of DNA binding sites by transcription factors
- By attracting transcriptional repressors to certain sites
