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Transcription
Biochem and medical genetics
| Question | Answer |
|---|---|
| RNA polymerase | Transcribes DNA into RNA in the 5' to 3' direction Can engage with DNA at specific sites and create sequences from scratch Base pairs RNA nucleotides to the template strand, allowing the RNA to be a copy of the coding strand |
| Differences between DNA and RNA | DNA - deoxyribose sugar, double stranded, contains thymine RNA - ribose sugar, single stranded, contains uracil |
| Direction of RNA synthesis | RNA polymerase synthesises RNA in the 5' to 3' direction Both strands of DNA can serve as templates, it depends on the direction of the gene You get different RNA depending on which strand is the template, so it is important the right strand is used |
| Different forms of RNA polymerase | RNA polymerase I - transcribes rRNAs RNA polymerase II - transcribes mRNA, snRNA, snoRNA and miRNA RNA polymerase III - transcribes tRNA and some snRNA These all have some structural similarities and some differences |
| Different RNA functions | mRNA - code for proteins rRNA - form the core of the ribosome and catalyse protein synthesis miRNA - regulate gene expression tRNA - serves as adaptors between mRNA and amino acids in protein synthesis snRNA - used in RNA splicing, telomere maintenace |
| Promotors | General transcription factors recognise specific sequences (promotors) located upstream of genes to be transcribed GTFs bind to the promotor sequences and recruit the correct RNA polymerase |
| General Transcription factors | Each polymerase as a set of associated GTFs that they require to be pulled towards DNA They provide enzymatic activity to unravel DNA Pol I - TF I x e.g. TFIA Pol II - TF II x e.g. TFIIF Pol III - TF III x e.g. TFIIIB |
| Core promotors | RNA polymerase is recruited to gene Core promotors The TBP subunit associates with the TATA box on the gene TFIIB joins by recognising the BRE sequence TFIIF recruits pol II and stabilises its interaction with TFIIB and TBP TFIIE and TFIIH join. |
| Role of TFIIH in transcription | Has helicase activity to unwind DNA Has kinase activity that phosphorylates the C-terminal domain of pol II to activate transcription |
| Other factors needed for RNA pol II transcription | In addition to GTFs many other proteins called activations and co-activators are required to ensure correct and regulated expression of genes These bind to regions outside the core promotor elements These ensure timely and controlled expression |
| Co-activators | indirect activation of pol II Use mediator proteins |
| Activators | Direct activation of Pol II Use gene specific transcription factors e.g. recognise sequence common in GTFs and regulate when they bind to the TATA box |
| Gene regulatory regions | Locus control region - regulates expression of a cluster of genes to ensure correct sequence of expression Enhancer - activates expression over long distances Silencer - represses transcription at promotors Insulator - regulate expression between genes |
| How do distant sequences affect transcription | DNA protein and protein-protein interactions Proteins create circular structures to being far away elements close together Allows proteins/elements at enhancers to be closer to their gene Can regulate ability of GTFs to associate with promotor |
| Heterochromatin | Tightly wrapped DNA regions containing inactive genes Cannot be accessed by GTFs Nucleosomes are tightly packed and associate with additional heterochromatin proteins bound to modified histone tails Histones are hypoacetylated and methylated at H3, K9 |
| Euchromatin | Loosely wrapped DNA regions containing active genes DNA can be accessed by GTFs Chromosomes are loosely packed with methylation of H4, K4 and K36 on histone tails. Acetylation also occurs |
| Modification of Histone tails | DNA must be accessible for transcription to occur. Modifications to histone tail N-termini play a key role in regulating this. Modifications like methylation and acetylation change the chromatin landscape of surrounding genes |
| Constitutive Heterochromatin | Mostly contains high copy number tandem DNA repeats Microsatellites spread through chromosomes' Centromeres made of satellite DNA Telomeres made of minisatellite DNA (long TTAGGG repeats) centromeres/telomeres are permanently heterochromatic |
| X inactivation in mammals | Xist expression on both X chromosomes in female ES cells is unstable. Stabilisation of Xist RNA acetylates, methylates, recruits proteins to histones and coats one X chromosome. The Xist on the active X is then silenced as one chromosome becomes inactive |
| Methylation of DNA | regulates gene expression High CpG methylation frequency Important for imprinting, transposon silencing and X-inactivation Cancers have changing methylation patterns Accessible chromatin unmethylated Methylated DNA reduces TF affinity |
| How does DNA methylation occur | When H3K4 is methylated on a histone tail, DNA methylating enzymes cannot bind so DNA remains unmethylated When this histone is non-methylated, the protein can bind and a methyl transferase methylates the promotor, inactivating it |
| Inactivating modifications | Methylated DNA Heterochromatin Methylated histones Tightly compact chromatin by binding of other proteins Organisation into nuclear lamina associated domains at the edge of the nucleus |
| Activating modifications | Unmethylated DNA Euchromatin Unmethylated and acetylated histones Chromatin held open by other proteins Organisation into transcription factories in the centre of the nucleus |
| Example pathway of activating a gene | Inflammatory cytokine binds to its receptor, triggering phosphorylation and activation of a TF This enters the nucleus and binds to an activator, recruiting other proteins and loosening DNA This then allows transcription of that gene |
| Consequences of CTD phosphorylation | Largest subunit of RNA pol II has a structure at its C terminus of 52 repeats of seven amino acids YSPTSPS Phosphorylation of the serine residues is critical for the transition between initiation and elongation in transcription |
| RNA polymerase II transcription cycle | Initiation - phosphorylated Promotor escape Promotor proximal pause - allows RNA manipulation Productive elongation - CTD phosphorylation triggers this stage Termination - cleaved to leave DNA |
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