Gene Regulation and Expression Flashcards
At which stages/levels can gene expression be regulated?
Chromatin level: chromatin modifiers, ie epigenetic factors, eg histone acetylation and methylation Transcription factors RNA Processing, eg Splicing mRNA Stability Translation Post-translational modification
Which enzyme is involved in the Transcription of mRNA?
RNA Polymerase II
What are control elements of a gene?
Segments of non-coding DNA that serves as binding sites for transcription factors
What are the two types of TFs?
Activators and Repressors
What are the 2 domains of an Activator TF?
DNA-binding domain
Activation domain: activates transcription
Outline the 3 ways in which Repressors can inhibit transcription.
- Directly binding to repression domain
- Indirectly: recruiting chromatin modifiers such as histone deacetylase and DNA methylases which lead to conformational changes to DNA thus silencing transcription
- By preventing Activators from binding
Explain Alternative RNA splicing.
Different mRNA molecules are produced from a single primary transcript by treating different RNA segments as exons and introns, which produces different mRNA products
Briefly explain RNA Interference
The phenomenon of inhibiting gene expression by RNA molecules.
miRNA and siRNA
How does miRNA inhibit gene expression?
By binding to nucleotides in the 3’ UTR of the pre-mRNA which results in the:
- degradation of pre-mRNA strand
- blocking translation, ie reduced translation
mature miRNA couple with RISC (RNA-induced Silencing Complex), which directly cleave mRNA or inhibit protein synthesis
Give 2 examples in which lncRNA inhibit gene expression.
Long non-coding RNA (>200bp), recently described
XIST RNA: produced from X-chromosome, coats X chromo to help silence/inactivate it.
HOX Antisense Intergenic RNA (HOTAIR): on HOXC locus, inhibit transcription through epigenetic regulation
What are the methods to analyse mRNA?
In-situ hybridisation Northern Blot semi-quantitative PCR quantitative PCR Microarray
Outline the process of In-situ Hybridization.
mRNA probe (cDNA specific to the mRNA) labelled with digoxigenin is added to the tissue/cells. Alkaline phosphatase-conjugated Ab is added, which binds to the dioxigenin. Colourless dye is then added which after removal of Pi by AP, it becomes purple and colours the cell
Outline the RNA extraction methods
- Gain access to RNA by disrupting/breaking cells using detergents
- Release RNA by denaturing proteins and nucleoprotein complexes
- Protect RNA by inactivating endogenous nucleases (by using chelating agents)
- Purify RNA: - extract using phenol/chloroform, then precipitate using ETOH
- Concentrate RNA
What are the main ingredients used for RNA isolation?
- Guanidium Thiocyanate (Trizol): lyse cells and prevent RNAse activity
- Proteinase K, B-mercaptoethanol, SDS: Degrades all proteins
- DNase I: degrades genomic DNA
- Phenol/Chloroform: separates aqueous and organic phases
- Isopropanol: precipitates RNA
