| 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 |
