Lecture 31 Flashcards
1
Q
RNAseq:
A
- Measure steady state levels of genes in the form of cDNA or RNA
- Can study gene structure
- RPKM: reads per kilobase per million reads
- You can determine what cells and what conditions produce the same reads, and can identify what genes are transcribed in what situation
- This can identify missed intron annotation, missed genes and exon branch sites depending on the condition
- Identifies gene boundaries nucleotide by nucleotide
2
Q
Transcription initiation is closer to gene regulation. Measure this with RNApol2 on DNA or using ChIP;
A
- Measure actual rates of transcription as this is the direct output of gene regulation
- Looking for the recognition of promoters by RNAP2
- A single gene can be studied over different time periods, to see when RNAP accumulates in a paused stance
3
Q
What did ChIP uncover in regard to RNAP2 binding?
A
- RNAP2 sits at the promoter waiting for another signal
- The TF recruits RNAP early, but it doesn’t transcribe, so it is ready to transcribe at very short notice
4
Q
Nuclear run-on assay (similar principle to ChIP):
A
- Transcription in vivo, but performed in vitro
- Lyse two cells on ice (one in the un-induced state, the other induced)
- Pellet nuclei
- Add NTPs (one radioactively labelled) and buffer to nuclei
- Incubate at 37 degrees for several minutes
- Isolate radio-labelled RNA
- The output is a reflection of the amount of transcription occurring in your gene of interest
- Only the transcripts that have already been bound by RNAP will transcribe, so the only ones we will pick up are the rounds of transcription that have already started
5
Q
Nuclear run-off:
A
- Similar experiment all performed in vitro
6
Q
ChIP:
A
- Cross link protein to DNA in living cells with formaldehyde
- Break open cells and shear DNA
- Add primary antibody of interest
- Add antibody binding beads
- Immunoprecipitate to enrich for fragments bound by protein of interest
- Reverse cross-links and treat with proteinase K (remove protein component, and left with DNA fragments bound by RNAP)
- Detect and quantify precipitate DNA through PCR and hybridisation methods
7
Q
eg) One gene, A, and want to know the occupancy of RNAP at different conditions:
A
- Primers will amplify up the fragments
- You know that your target DNA under non-induced conditions will give you a small band (little bit of RNAP)
- Under induced conditions you will get a larger band, as there will be more RNAP bound at the promoter
- This is a measure of transcription rates, because it is looking at RNAP sitting at the promoter
8
Q
ChIP can be performed on a genome wide scale:
A
- Do the same experiment using sequencing libraries, remove the PCR step and replace with a next generation sequence
- Make a sequencing library that you can run on a platform
- Measure the level of every fragment RNAP is bound to
- A density of map is produced with the density of reads across the genome
- Complements RNAseq, but looking at specific elements of the transcription process
9
Q
Transcriptional fusion:
A
- Reporter genes (gfp, or GUS etc) are fused to promoters of gene of interest
- Indirect measure of transcription by measuring reporter gene product
- Qualitative cell or tissue specific expression, but not quantitation
- Which parts of the promoter are important for the expression of your gene of interest? (slowly chop down the promoter region)
10
Q
Protein abundance and protein function:
A
- Wester blots, immunoprecipitation
- Enzyme activity and immunfluoresecence and epitope tags
11
Q
Proteomic approach:
A
- Separate total protein content by two dimensional gel electrophoresis, identify proteins by mass spectrometry
- ## This is two dimensional separation of proteins
12
Q
Protein interaction studies:
A
- Proteins usually operate in complexes
- The gene of interest is translationally fused to TAP-tag, purify and then detect
- Calmodulin beads pull out the interacting factors as a pure protein complex
- Mass spec will determine exactly what proteins are acting
- Temporal and spatial investigation
13
Q
Gene expression in whole organisms:
A
- Tagging proteins in the embryo can allow us to understand proteins involved in cell development over time
- Spatial changes in gene expression related to localisation and specification
14
Q
Measuring expression in the context of regulation:
A
- DNA to RNA to the cytoplasm where proteins are expressed that perform a specific action.
15
Q
Gene product function can be controlled:
A
- Through activation based on location (cytoplasm vs nucleus)
- Processing, degradation, targeting etc
