How does Electrophoretic mobility shift assay work?
needs a source of purified protein and a labelled target sequence.
mix target DNA and activator on a non-denaturing gel
How do Reporter Assays works?
- transfect the cells of interest with 2 plasmids
- one plasmid contains the gene that encodes the transcription of interest = will be expressed
- the other plasmid has a reporter gene with a binding site of interest.
How does Chromatin Immunoprecipitation (ChIP) work?
add crosslink proteins to DNA
- activated protein is bound to its site on DNA and stuck there
- cells are broken up and DNA sheared
- there is a mixture of DNA bound with protein and other uninterested bits of DNA
- purify the sequences bound to the activator and use an antibody that is specific to the activator to purify the activator bound to its site.
- reverse the crosslinks and remove the protein
How do activators actually work?
- Promote binding of an additional activator
- Stimulate complex assembly (RECRUITMENT)
- Release stalled RNA polymerase (stimulate activity) = heat shock genes such as hsp70
How is H3lys9me heterochromatin assembled?
- lysine is deacetylated
- methylation of lysine9 is mediated by an enzyme called Suvar 39 on H3
- the methyl mark is recognised by a croma domain proteins called heterochromatin protein1
- HP1 is assembled onto chromatin
How does X chromosome inactivation occur?
- both X chromosomes are active during early development = express T6 transcript
- Later in development one of the X chromosomes stops expressing T6 and start expressing Xist which moves in the other direction.
- Xist starts to coat the X-chromosome and recruit H3k27 methylase
- recruits further silencing factors
How is NF-kappaB activated?
- NF-kB complexes are held in inactive complexes in the cytoplasm
- TNF binds to receptor complexes on membrane, it activates the IKK complex
- IKK complex phosphorylates IkB which then signals the ubiquitination process
- NF-kB is no longer bound to its inhibitor and is translocated into the nucleus of the cell where it binds to its target sequences in the enhancers of the gene.
What happens in translation initiation?
- methyanine is the initiator tRNA and spans to eIF2 to form a tertiary complex
- small subunits starts scanning and scans until AUG start codon
- eIF2 partially leaves, rest of initiation factors leave and eIF5B is bound to GTP and release of eIF5B and eIF1A
What happens in mRNA degradation phase I?
- decaying enzymes remove the cap and open up the 5’ end of the RNA
- deadenylases = remove the polyA tail from the 3’ end, and the mRNA can be degraded
- endonuclease = cut the mRNA in the middle = 2 open ends that can be degraded
What happens in mRNA degradation phase II?
Phase 1: cap and PolyA tail are removed
Phase 2: exonuclease degrades the RNA from the open ends
What is the mechanism of siRNA/miRNA function?
- cut by dicer to produce double stranded RNA
- double stranded RNA is unwound into the RISC complex
- RNA binds an argonaute protein
- siRNA forms enzymatic complex where the siRNA is the substrate binding part
- miRNA = assembly of RISC complex but its not an endonuclease
- leading to the translation block and the turnover of mRNA
