Transcriptional Control Flashcards
1
Q
Eukaryotic RNAP
A
- 3 kinds
RNAPI: rRNA (nucleolus)
RNAPII: mRNA
RNAPIII: tRNA, small RNA
2
Q
Bacterial vs. Eukaryotic RNAPs
A
- structural ortholog elements
- however, eukaryotic RNAP cannot bind in a sequence specific way on their own
- require transcription factors to recognise promoter and form bubble
3
Q
Eukaryotic Basal Transcription
A
- need to initate transcript
- assembled initation complex identical for every RNAPII gene giving low levels of unregulated transcription
- further gene specific TF give regulated levels of expressoin
4
Q
Eukaryotic Promoter Model
A
- proximal gene specific TF
- distal gene specific TF (loop around to interact with start site basal initiation complex)
5
Q
TATA Box
A
- 25/-30 upstream from gene
- recognised by TFIID
- stronger binding with higher sequence specificity
6
Q
TFIID
A
- multiprotein complex of TATA binding protein and other subunits
- binds sequence specifically to TATA box
- binding stabilised by TFIIA/TFIIB
- TATA binding protein kinks DNA
7
Q
Electrophoretic Mobility Shift Assay
A
- shows how TFIIB stabilises binding
- with presence of TFIIB you get strong binding of TBP to the DNA
- bending DNA is energetically inefficient
8
Q
Other Promoter Elements
A
- initiator element (+1)
- downstream promoter element (+28/+34)
9
Q
Additional Basal Factor
A
- TFIIF binds RNAPII in solutoin and delivers it to TFIID/B/DNA on the promoter
- TFIIE and TFIIH responsible for 3 critical functions in transcription: phosphorylation of RNAPII, promoter melting, promoter clearance
10
Q
Phosphorylation of RNAPII
A
- C terminal tail of 26 repeats of a 7 residue sequence phosphorylated by TFIIH kinase
- Hypophosphorylation: CTD associated with intitiation complex
- Hyperphosphorylation: CTD associated with elongation competent RNAPII
- regulates 5’ capping, assembly of spliceosomes, binding of cleavage/polyadenylation complex
11
Q
Promoter Melting
A
- TFIIH melts dsDNA around start site to form bubble
- bubble unstable and if initiation doesn’t occur in this time span ATP hydrolysis is needed
- regulates rate of initiation of transcription
12
Q
Regulatory Regions
A
- in addition to promoters, eukaryotic genes contain binding sites for gene specific TFs
- sequence specific binding of gene specific TFs allows genes to achieve and maintain controlled levels of tissue specific expression patterns
13
Q
Gene Specific TF
A
- eukaryotic factors usually transcriptional activators
- bind to subset of genes through sequence specific DBD
- modulate activity of promoter bound transcriptional machinery through activation domains
14
Q
DNA Footprinting
A
- use DNaseI to cleave sequence of DNA bound with and without protein
- with protein will show gaps in blotting assay where nuclease cannot cut protein bound DNA
15
Q
DNA - Protein Interactions
A
- electrostatic bonds (long range)
- hydrogen bonds
- VDW forces
- hydrophobic interactions
- structural complementarity
16
Q
Sequence Specific Interactions to DNA
A
- electrostatic interactions provide stabilising energy but not specificity
- TF binding doesn’t cause unwinding
17
Q
B DNA
A
- antiparallel right hand double helix
- minor and major groove
- bases project into interior
18
Q
Groove Contacts
A
- absolute recognition of the four different base pairs is only possible via the major groove
- asymmetric hydrogen donor acceptors (G:C)
- bulk methyl group on T residues
- minor groove binding can only distinguish A:T from G:C (not flipping of each pairing)
19
Q
Helix Turn Helix
A
- two alpha helices separated by a loop
- recognition helix (major groove), N terminal helix
- side chains of recognition helix hake base specific contacts
- form dimers to recognise palindromic seuqneces
20
Q
Leucine Zipper
A
- cJUN/cFOS heterodimer
- encoded by separate genes (oncogenes)
- bind to form Y shaped DBD
- binding of helices to form Y shape DBD involves hydrophobic interactions between regularly spaced leucines on both helices
- forms a tight leucine zipper
21
Q
Helix Loop Helix
A
- cMAX can form homodimer or heterdimer with cMYC
- complex version of leucine zipper containing loop interrupting a helixes
- binding of helices to for Y shape still involves hydrophobic interactions between regularly spaced leucines on both helices
22
Q
Zinc Finger Domains
A
- zinc atom coordinated by cysteine or histidine
- no bacterial
- B turn and a helix held together by coordinated ZN atom (4 residues)
- work in tandem with each finger recognising a 3 nucleotide target site in DNA
23
Q
p53 DBD
A
- tumor suppressor causing apoptosis
- central domain binding DNA in a sequence specific manner
- no structural similarity to any other known DBD
24
Q
Activation Domains
A
- stimulate activity of basal transcriptional machinery
- unusual amino acid composition characteristic of IDPs
- no defined 3D structure: high charge density and prolines prevent this
- versatile binding to many targets
- aid recruitment of basal factors, create open chromatin, stimulate enzymatic activities
25
Q
Partial Nuclease Digestion
A
- micrococcal nuclease digest linker DNA between nucleosomes
- generation of distinct DNA fragments containing one or more repeat units
26
Q
Nucleosomes
A
- 4 core histones: H2A, H2B, H3, H4
- all four histones contain central histone fold (dimerization motif)
27
Q
Histone Dimerization
A
- tight binding with electrostatic and hydrophobic forces
28
Q
Histone Octamer Assembly
A
- 2 copies each of H4/H3 form H3-H4 tetramer
- H2A-H2B dimer forms
- two components brought together to form the histone octamer
29
Q
Histone Evolution
A
- highly conserved in eukaryotes
- probably emerged early during evolution
- likely they evolved to stabilize and protect DNA in extreme conditions of euryarchaeota
30
Q
Nucleosome DNA Interactions
A
- rigid DNA bent around nucleosome unfavourably in two tight circles due to interaction strength
1. ionic/H bonds (histone positive)
2. non polar contact
3. intercalation of arginines to phosphates across the groove
4. amide phosphate bind interactions
5. histone helix dipoles
31
Q
DNA Distortion
A
- major groove widened to prevent TF binding
- recudes spatial complementarity
32
Q
Linker Histones
A
- H1/H5 stabilise interactions between nucleosomes in compacted chromatin
- organise entry and exit ponits of DNA
33
Q
Chromatin Organisation
A
- DNA wrapped around histone octamer as nucleosome
- nucleosomes held together by H1 linker protein
- forms chromatine fibers
34
Q
Solenoid
A
- The solenoid structure of chromatin is a model for the structure of the 30 nm fibre. It is a secondary chromatin structure which helps to package eukaryotic DNA into the nucleus.
- can be open or compact
35
Q
Disproving Solenoid Model
A
- doubt about validity of model in vivo
- chromEMT saw irregular patterns of DNA and no evidence of solenoids
- still have looser and tighter conformations
- concluded that nucleosomes assemble into disordered chains with different arrangements
- little evidence for regular helical structure
- chains are flexible and bend at various length to achieve levels of compaction
36
Q
Histone N Termini
A
- N termini sequences control gene regulatory properties
- highly flexible
- emerge from nucleosome
- acetylated by conserved lysines
- compact to interact with backbone
37
Q
Histone Acetylation
A
- acetylation of N termini creates open chromatin
- exposed DNA closer to feed into RNAP from nucleosome
- opens dna
- deacetylation allows positively charged N termini to bind DNA on neighbouring nucleosomes
38
Q
Chromatin Opening
A
- gene specific TFs recognise target sequence in compacted chromatin
- recruit other enzymes
- localised opening/closing
39
Q
Methylation
A
- effect depends on position/residue on histone
- nucleosomes in heterochromatin usually methylated
40
Q
c-Myc
A
- proto oncoprotein
- 60-70% of cancer dependent on mutation
- is a TF
41
Q
c Myc Function
A
- regulates expression of genes involved in cell proliferation
- increases metabolic rate
- increased nutrient uptake, enhanced glycolysis, etc
42
Q
c Myc Overexpression
A
- normal WT protein but translocation to another chromosome under the control of active immunoglobin H promoter
43
Q
c Myc stability
A
- short WT half life
- stability of cMyc increased between due to the presence of mutations in particular regions of the proteins that initiate its degradation
- prevent degradation supporting growth of cancers
44
Q
c Myc DBD
A
- helix loop helix motif
- associates with cMax with HLH DBD
- positively charged arginine residues contact negatively charged phosphates on DNA
- hydrophobic leucine form leucine zipper
45
Q
c Myc Structure
A
- intrinsically disordered aside from HLH region
- transcriptional activation domain
- N termal IDP can interact with large number of TF in specific by conformationally flexible manner
- conserved residues tend to be hydrophobic and bulky
- islands of low homology
- transient secondary structure and modelling with molecular dynamics simulations
