Cell Nucleus II Flashcards
transcription cycle of bacterial RNAP
- holoenzyme assembles, finds promoter
- polymerase unwinds DNA at transcriptional start site
- initial transcription - abortive initiation - is inefficient, short transcripts are released.
- once ~ 10 nts have been synthesised, interactions with promoter DNA are broken, the sigma factor is released and polymerase tightens around DNA and shifts to processive elongation mode.
- transcribed RNA is released when a termination signal is reached.
what is TATA recognised by
TBP - TATA binding protein. part of TFIID
what is DPE
downstream promoter element
what is the initiator
Py2CAPy5
conserved at the transcriptional start sequence
what forms the pre-initiation complex in transcription
assembly of transcription factors and RNAP on DNA
what do GTFs carry out an equivalent function to
bacterial sigma
TFIID consists of
TBP and TAFs
TFIID
Recognise TATA box, then starts assembly of other GTFs eg TFIIB
TFIIA
Stabilises interaction of TFIID with DNA
TFIIB
Bridges TFIID and Pol II Binds BRE (TFIIB recognition) element of promoters, positions RNAP at the transcription start site
TFIIF
Stabilises interaction of RNAP with TBP and TFIIB
TFIIE
Enters after TFIIF, attracts TFIIH
TFIIH
Has helicase activity so unwinds DNA at the transcription start site
Phosphorylates Ser5 of RNAP CTD
Releases RNAP from promoter
preinitiation complex assembly order (transcription)
TFIID TFIIA TFIIB TFIIF + RNAP TFIIE TFIIH
PolII CTD control
No phosphorylation of Pol II before binding DNA
TFIIH phosphorylates Ser5 upon RNAP binding, helps with 5’ capping
Phosphorylation of Ser2 activates elongation, splicing, polyadenylation
Dephosphorylation leads to release and recycling of Pol II
3 actions of seq specific TFs
- direct gtf recruitment
- indirect gtf recruitment
- changes in chromatin structure
mediator
a coactivator, interacts with pol II CTD. many transcriptional regulators act via.
regulation of regulators (2)
modulate levels of factor
modulate TF intrinsic activity
modulating levels of a factor
- tissue specific expression
- identify with radioactive probes on cell extracts or RNA FISH to look at mRNA levels in living tissue
Modulation of TF intrinsic activity
Ligand binding: eg steroid hormone receptors
Glucocorticoid receptor: hormone binding causes conformational change in receptor, inhibitory protein is released and NLS exposed. GR translocates into nucleus, binds target genes.
For other TFs, they are already present in the nucleus and ligand binding activates binding properties.
control of TF activity by phosphorylation
eg p53
p53 normally unstable, after DNA damage it becomes phosphorylated by ATM and chk2.
This dissociates mdm2, a negative regulator
Mdm2 normally targets p53 for proteasomal degradation (Mdm2 is an E3 ubiquitin ligase)
After DNA damage, p53 levels rise; p53 binds target genes so activates transcription ⇒ eg cell cycle arrest or apoptosis
histone acetylation
- By histone acetyl-transferases (HATs)
- Generally promotes transcription, chromatin opens up as +ve charge neutralised
- Histone deacetylases HDACs remove acetylation - represses transcription
RB and E2F: control of transcription via chromatin modulation
- Defective in retinoblastoma cancer
- RB binds and inhibits E2F: a seq-specific regulatory factor
- RB recruits HDACs ⇒ transcr repression
- RB phosph in S phase, dissociates from E2F
- E2F can now bind HATs, induction of transcr of S-phase specific genes.
thyroid hormone receptor
Without thyroid hormone (ligand), HDAC binds TFs, repressing transcription.
Binding of ligand to receptor causes HDAC to dissociate, HAT activity is stimulated and transcription is activated.
DNA footprinting
- 32P label one strand of dsDNA, sep into 2 tubes
- Add binding protein to one sample
- Perform limited nuclease digestion with DNAse I on both samples (so that only cuts once)
- Analyse sizes of resulting cleaved ssDNA (labelled bands) by autoradiography
- Compare two reactions on a denaturing gel to find the protected (binding) site - will appear as gap in ladder of all other fragment sizes
ChIPseq
- Crosslink chromatin and DBPs in live cells using formaldehyde
- Shear DNA into small fragments
- Purify TF-DNA complexes with TF specific antibody (magnetic beads linked to antibody)
- Amplify DNA with PCR
Sequence, map reads to genome to locate binding sites
Evolution - DNA vs proteins
Do cis elements or TF structure change to change regulation?
cis elements (DNA) more quickly evolving,
eg down’s syndrome mouse: has human chromosome 21 added. For liver TFs, DNA binding pattern is same same as in human liver for the human chromosome, DNA wins.
enhancers
DNA loops back so enhancers come into contact with promoters
insulators
regulate DNA looping - CTCF insulator TF binds DNA, interacts with cohesins and cause changes in DNA looping. Eg can insulate genes from enhancer elements
Examining DNA looping experimentally - using HiC
position effect variation
chromatin state inherited through mitosis and cell division. Eg constitutive heterochromatin is maintained stably (centromeres)
Difficult to maintain precise boundaries between euchromatin and heterochromatin
PEV drosophila example
- Fruit flies - translocation of white gene to boundary between euchromatin and heterochromatin (genetic engineering) ⇒ silenced in some cells but not others
- Normal function of the White gene provides red colour in cells of the eye. Loss of White expression results in white patches.
- Larger patches of white or red in the eye suggested that once the White gene is either expressed (euchromatin) or not expressed (heterochromatin) in a given cells, the daughters of this cell maintained this state ⇒ epigenetic inheritance through mitosis
- Initial setting of the chromatin boundary is random, but once set it can be maintained.
Polycomb - memory of body plan in drosophila
((maintain Hox gene expression even after initial transcriptional regulators disappear))
- initial body plan info: spatially restricted expression of Hox gene TFs
- enhancer - binds TFs early in development, provides positional info
- polycomb acts through PRE (polycomb response element) maintaining the memory even without the TFs
- acts through modification and maintenance of chromatin structure
DNA methylation
- at CppG motifs, can maintain as symmetric
- DNMT1
remove by oxiation - TET
imprintingsh
mammals
- different methylation patterns of maternally and paternally inherited alleles at fertilisation
IGF2: expressed in placenta as imprinted gene, important for foetal development (increases placental size and nutrient transfer)
Copy switched on in placenta = always from father, evolutionary arms race theory. Father wants as much of the mother’s resources as possible to go into the baby, mother wouldn’t want to put all resources into baby as will influence lifetime reproductive chances.
miRNAs
dicer processes primary transcripts, gives short ss miRNA
bind argonaute proteins
bind 3’ UTR of target mRNA, inhibits translation
siRNAs (RNAi)
long dsRNA processed by dicer into short siRNAs
bind argonaute (RISC)
passenger RNA strand removed and degraded
guide RNA binds target mRNA
Argonaute slicer activity cuts up target RNA
piRNAs
expressed in germline, protection from transposable elements
long ncRNA
Xist: role in X inactivation in mammals. Coats over inactive X to form barr body
