genetics lecture 1 - regulation of transcription 1 Flashcards
what does each gene have?
Each gene has its own requirements for regulated expression
how can gene expression be regulated?
DNA –> rate of transcription
RNA –> mRNA processing and stability
+ Rate of translation –> Protien = Protein folding, modification, and stability
what are marker genes and specific examples of them?
Marker genes can indicate which cell types are present
ES cells –> OCT4 - Transcription factor for self renewal
cardiomyocytes –> Cardiac troponin C (TNNC1) - Binds Ca2+ to activate muscle contraction
neurons –> MAP2 - Stabilises microtubules in dendrites
What method can we use to quantify mRNA expression?
quantitative RT-PCR / real time RT-PCR
A technique for amplifying and quantifying the amount of a specific RNA present in your sample
q = quantitative / real time
RT = reverse transcriptase
PCR = polymerase chain reaction
what are the Steps to quantify mRNA expression?
1 - Culture cells and treat them according to your experimental protocol
2 - Purify RNA from the cells
3 - Reverse transcriptase reaction to synthesise cDNA from RNA
4 - PCR: polymerase chain reaction
what is the Reverse transcriptase reaction to synthesise cDNA from RNA step?
Taq polymerase cannot use RNA as a template.
Need to convert RNA to cDNA first.
Key reaction components:
reverse transcriptase enzyme (RT)
oligo(dT) or random hexamer primers
dATP, dTTP, dGTP, dCTP
buffer
high quality RNA
what are the stages of PCR?
Step 1: denaturation at 92°C
Step 2: annealing at 60°C
Step 3: extension at 72°C
Repeat cycle up to 30 times
-Each cycle amplifies the DNA by two fold.
-Total amplification after 30 cycles is in the millions
-Very sensitive, so contamination with other DNA must be avoided
-Specificity determined by primer sequences
Advantages: Rapid, Sensitive, Accurate
what is Semi-quantitative PCR?
Agarose gel electrophoresis after a set number of cycles.
Detect by DNA stain. Semi-quantitative
what is Real time / quantitative PCR?
SYBR green fluorescent dye intercalates into double stranded DNA products
PCR products are quantified at every cycle
High precision and high throughput.
Large dynamic range
what is the Quantitative relationship between amount of starting material and amount of PCR product?
Threshold= arbitrary level of fluorescence
Ct= PCR cycle number at which fluorescence becomes greater than the threshold.
Quantification calculations performed using the Ct for each reaction, normalised to the reference gene.
what is a Reference Gene?
a reference gene is aimed to normalise possible variations during:
- sample prep and handling (use the same number of cells from a start)
- RNA isolation (RNA quantity and quality)
-reverse transcription efficiency across samples/ experiments
- PCR reaction set up
- PCR reaction amplifies efficiencies
Reference / control genes are chosen that do not change during the experiment.
Often used: β-actin, Gapdh, βIII-tubulin
how is Quantitative RT-PCR is performed and what determines the volume of marker gene needed?
Quantitative RT-PCR is performed using primers for the marker genes, and for the reference/control gene GAPDH.
The quantity of each marker gene is normalised to the quantity of the reference gene, so as to calculate the relative mRNA expression.
Using two dishes of cells, you try to differentiate ES cells into neuronal and cardiomyocyte cells in your lab.
What will you need to do next, in which order, to determine whether your differentiation was successful?
purify RNA from the cells, make cDNA, perform real time PCR
You’ve made the cDNA. What reagents will you need for the real time PCR step?
-thermostable polymerase and its buffer
-primers for a reference gene
-SYBR green dye
-PCR machine with fluorescence capability
-primers for OCT4, cardiac troponin C (TNNC1) and MAP2
what are the 3 types of RNA?
ribosomal RNAs (rRNA), RNA polymerase I 80% of RNA
messenger RNAs (mRNA) RNA polymerase II 5% of RNA
transfer RNAs (tRNA) RNA polymerase III 15% of RNA
What is EGF?
A growth factor that binds to a cell surface receptor
what is elk1?
A transcription factor that binds to DNA
what are the main stage of transcription?
initiation =
1 - polymerase binds to promoter sequence in duplex DNA “closed complex”
2 - polymerase melts duplex DNA near transcription start site, forming a transcription bubble “open complex”
3- polymerase catalyses phosphodiester linkage of 2 initial rNTPs
elongation =
4 - polymerase advances 3 –> 5’ down template strand, melting duplex DNA and adding rNTPs to growing DNA
termination =
5 - at transcription stop site, polymerase releases completed RNA and dissociated from DNA
what are transcription factors?
Initiation of RNA pol II transcription is regulated by transcription factors
Transcription factors are proteins that binds to specific DNA sequences
Transcription factors recruit co-activators and RNA polymerase II
what are Gene regulatory elements?
Transcription factors bind to specific sequences in gene promoters
Enhancers can be 100s of kb away from the promoter, and can be downstream of the initiation site.
They are thought to loop over and interact with the factors bound at the promoter
Basal/general transcription factors
Found at most pol II genes
Recruit and activate pol II
how are genes regulated?
by combinations of several different transcription factors
Transcription factors have a modular structure
an Activation domain (AD) bonded to an Activation domain (AD)
where do transcription factors bind?
co-operatively at the promoter of a gene.
how is are basal transcription factors recruited?
The TATA box recruits TFIID, which consists of TATA-binding protein (TBP) and TBP- associated factors (TAFs).
TBP binding induces a sharp bend in the DNA
TFIID and nearby sequences recruit other basal or general transcription factors
what is The pre-initiation complex (PIC)?
Transcription factors bind to specific DNA sequences and recruit
co-activators, in particular the mediator complex, which is required for nearly all RNA polymerase II-transcribed genes
The mediator complex acts as a bridge between TFs, basal transcription factors and the C-terminal domain of RNA pol II
Assembly of the pre-initiation complex allows phosphorylation of the polymerase C-terminal domain (CTD)
CTD phosphorylation allows the RNA pol II to clear the promoter and initiate transcription
how do you regulate transcription factor (TF) activity?
1) Activating a transcription factor by altering its conformation through ligand binding or phosphorylation.
The conformational change can:
Alter its ability to bind DNA
Alter its ability to interact with other proteins
Alter its nuclear localisation
2) Altering the expression level of a transcription factor
3) Altering the accessibility of the DNA binding site to regulate transcription factor binding
How can transcription factor activity be regulated?
1 - Phosphorylation-activated DNA binding
2 - Nuclear localisation regulated by ligand binding or phosphorylation
3 - Ligand-activated DNA binding
4 - Ligand or phosphorylation-dependent co-factor recruitment
what is the MAPK pathway – Mitogen activated protein kinase pathway
1 - Growth factors bind to cell surface receptors
2 - Receptor activation sets up cascades of protein phosphorylation inside the cell, amplifying the signal.
3 - At the bottom of the cascade, transcription factors get phosphorylated and activated, driving transcription.
MAPK signalling can drive proliferation, or stress responses
Oncogenic mutations that constitutively activate MAPK signalling are common in cancer
what drugs that target MAPK signalling used in cancer treatment
Herceptin (breast cancer)
Gefitinib (lung cancer )
Trametinib (melanoma)
Vemurafinib (melanoma)
how are EGF signalling leads to Elk1 phosphorylation and DNA binding?
EGF –> binds to EGF receptor on cell membrane –>which activated Ras/Raf –> this phosphorylates MEK –> this phosphorylates ERK which phosphorylates Other TFs and co-factors and Serum response element (SRE)
eg regulating c-fos gene expression
what is SRF?
Serum response factor (SRF) core binding site: CCATATATGG
Core binding sites are typically 4 – 12 bp long; often palindromic
