Gene Expression Flashcards
Three domains of a transcription factor
DNA binding domain
Activation domain
Ligand binding domain
Three stages of transcription
Initiation
Elongation
Termination
Transcription factor categories based on DNA binding/dimerisation domains
Helix loop helix - myc Lucine zipper - AP 1 Zinc finger - steroid receptor Helix turn helix Homeodomain (helixloophelixturnhelix) - Hox
Activation of transcription factors can be constitutive or regulatory. What are the subsets of regulatory?
Developmental(cell specific)
Signal dependent -> steroid receptors, internal signals, cell surface receptors.
Transcription factors bind..
Response elements in promotor region
TFs function by:
Stabilizing preinitiation complex
Recruiting co-activators
Altering chromatin
[can be negative as well]
Three tests that measure gene expression
Microarray
Northern blot
qRT-PCR
Tests to study gene expression
Promotor reporter assays Nuclear run on assays Binding assays In vitro > EMSA, DAI In Vigo > Chromatin immunoprecipitation
Steps followed when doing EMSA
- Obtain nuclear extract
- Incubate with radiolabelled probe of binding domain
- Run on PAGE
- Develop and detect on film
Steps to microarray
- Isolate mRNA
- Generate cDNA (RT)
- Label the two samples with different probes
- Hybridise onto assay
- Imaging
Steps of capping and methylation
- Gamma phosphate hydrolysed off 5’ triphosphate of mRNA (phosphohydrolase)
- Guanylyl transferase covalently joins GTP to RNA chain
- Methyl transferase methylated guanine at the N-7 position
- Can be further methylated CAP0>CAP1>CAP2
Capping catalysed by three enzymes
Phosphohydrolase
Guanylyl transferase
Methyl transferase
Why have a cap? (And polyA tail)
Promotes stability of mRNA
Prevents degradation by 5’ exonucleases
Promotes translation of mRNA
Helps to recruit mRNA to ribosome
Two steps of polyadenylation
Cleavage: RNA cut 10-30 nucleotides down from AAUAAA
Addition of As: 100-200 added (no template needed)
What adds polyA tail
PolyA polymerase
Sequence of branch site in intron
YNYRAY
Y = pyrimidine R = purine N = anything
What forms the spliceosome?
snRNP
Pre-mRNA
What makes up snRNPs
snRNA - small 100-200 ntds, rich in Us
10 different proteins
Importance of snRNAs
Mediate sequence specificity/accuracy
Fold into 2ndry structures - stem loops
Catalyse splicing
Central outcome of alternative splicing
One gene can produce multiple mRNAs and proteins
Additional RNA processing events
Trans splicing of exons from different mRNAs
mRNA editing
Transport to cytosol
RNA degradation in eukaryotes
- Shortening of PolyA tail
- Removal of 5’ cap
- 5’ to 3’ exoribonuclease
RNA instability elements
ARE elements - A/U rich elements
Steps in microRNA pathway
The miRNA fold back on itself Enzyme DICER cuts it into short segments One strand of dsRNA degraded Other strand complexes with proteins Complex can target any complementary sequence Prevents gene expression
Stages of translation
Assembly of initiation complex AUG
Subunits of ribosome binds
Translation begins
Elongation of polypeptide chain
Examples of translational control
MicroRNA
Iron response element binding protein
RNA masking
Post translational modifications of proteins
Phosphorylation/dephosphorylation Ligand and co-factor binding Protein protein interaction Protein degradation Peptide cleavage Etc
Applications of microarray
Track gene expression patterns associated with disease
Identify gene as diagnostic markers/ therapeutic targets
Identify changes in gene expression in response to targets
Why prepare cDNA from mRNA
More stable
Used to quantify expression levels -> RT-PCR
Used for expression profiling , mircoarrays
