BN - Transcriptional Regulation II Flashcards
Where does transcription occur in prokaryotes and eukaryotes?
In prokaryotes, transcription and translation occur in the same compartment
In eukaryotes, transcription is purely in the nucleus and translation in the cytoplasm
- This allows greater degree of control over these proceses
What is comprised in the C-Terminal Domain and what is its role in Elongation?
CTD
- 52 tandem repeats of 7 amino acids
- Each contains 2 serines (Ser2 & Ser5)
- Phosphorylated
C-Terminal domain (CTD) of RNA Pol II phosphorylated
- Phosphorylation allows loading of RNA processing machinery
What is the importance of Post-transcriptional processing?
The RNA transcript has to be tagged and modified to identify it as mRNA that will ultimately be used to produce protein
What are the 5 main steps involved in Post-transcriptional processing?
- Addition of 5’ Cap – protection and marking a mRNA
- Splicing - to remove non-coding regions
- 3’ processing and polyadenylation – marking as the end of the mRNA and protection
- Editing (rare but important) – similar to mutating the mRNA to produce alternative final product but this is not a random event
- Transport – to cytoplasm for translation.
What is involved in the Addition of the 5’ Cap?
- The 5’ cap is guanosine with a methyl group on the 7-position (m7G)
Essential for efficient translation, stabilisation and transport of mRNA
- The first thing that happens to the RNA is addition of a 5’ cap
- Addition of 7 methyl-guanosine to the 5’ end of RNA
- The RNA is capped as soon as it emerges from the exit channel of RNA polymerase II (~25-30 bp)
Capping enzymes bound to the RNAP II’s C-terminal domain (CTD).
The first base at 5’ end of the RNA contains three phosphates:
alpha, beta, gamma
What are the 3 detailed steps and enzymes involved in 5’ cap addition?
1. RNA triphosphatase removes the gamma-phosphate
GMP is added to the 5’ end of the RNA
2a. Guanylyltransferase
- Removes the gamma and beta phosphates of GTP
- GMP (guanosine monophosphate)
2b. Guanylyltransferase
- Then the GMP (guanine) is added to the 5’ terminal base of the transcript
G is added to the RNA in reverse orientation from all other nucleotides forming a 5’-5’ linkage
3. Methyltransferase
- Adds methyl group to guanine (7th pos of the purine)
Why is the addition of a 5’ Cap important? (3) and why is properly capped RNA necessary?
- The cap helps to distinguish mRNA from other RNA
- Helps mRNA be properly processed and exported from the nucleus
- Protects it from degradation in the cell
Improperly capped RNA is recognised by quality control mechanisms and becomes degraded
Polycistronic vs Monocistronic
Prokaryotes
- Polycistronic: mRNA encodes two or more proteins, no introns
Eukaryotes
- Monocistronic: mRNA encodes a single protein, intronic
What are 3 classes of RNA splicing?
Nuclear pre-mRNA splicing
Most eukaryotic genes (common)
- Two transesterification reactions (branch site A)
- Spliceosomes
Group II self-splicing
Some eukaryotic genes (rare)
- e.g. rRNA, organellar mRNA or fungi, plants and bacteria
- Two transesterification reactions (branch site A)
- RNA enzyme encoded by intron (ribozyme)
Group I self-splicing
Some eukaryotic genes (rare)
- e.g. introns in eukaryotic viruses
- Two transesterification reactions (branch site G)
- RNA enzyme encoded by intron (ribozyme)
What occurs in Step 1 of the 2 transesterification reactions?
Step 1 : cleavage at the 5’ splice site
- The backbone hydroxyl group (OH) of the A within the branch point acts as a nucleophile and attacks the 5’ splice site
- Intron folds back on itself
- Phosphodiester bond between A (BP) and G (5’SS)
What occurs in Step 2 of the 2 transesterification reactions?
Step 2: cleavage at the 3’ splice site and joining of the exons
- The newly liberated 3’OH group of the 5’ exon becomes a nucleophile and attacks the phosphoryl group at the 3’ splice site
- Cleavage at 3’ splice site
- Exons ligated together
- Intron released and degraded in cell
