RNA processing

Question 1

Transcription and translation - coupling or compartmentalisation

Answer 1

In E.coli
- Transcription coupled to translation
- Before transcript finished, polypeptides can bind and start protein synthesis
- Allows rapid gene expression
- Coupling
In eukaryotic cells
- Transcription ~ nucleus
- Translation ~ Cytoplasm
- Slower but allows regulation during gene expression, gives extra protection
- Compartmentalisation

Question 2

Eukaryotic mRNAs processed in nucleus

Answer 2

Eukaryotic mRNA processing - 3 main steps
1. Capping at 5’ end
2. Removal introns (pre-mRNA splicing)
3. 3’ end processing (cleaving) and polyadenylation
Modification occurs through entire process
Cleaving - as RNAP moves cleaving occurs releasing mRNA
Polyadenylation - adding a poly(A) tail to 3’ end

Question 3

The “M^(7)G cap” structure of eukaryotic mRNA

Answer 3

Universal - all eukaryotic transcripts receive it
G nucleotide added to 5’ end RNA pol II transcript
Cap linked by 5’-5’ triphosphate linkage, unique linkage and not susceptible to cleavage
Cap protects RNA structure from degradation
Cap nucleotide is methylated
First transcribed nucleotide often modified

Question 4

Eukaryotic mRNAs are monocistronic transcripts

Answer 4

In prokaryotes
- Protein coding structures organised by operons
- Has capacity to be translated into multiple different functionally related proteins
- Automatically upcodes
In eukaryotes
- In monocistronic
- Encodes single polypeptides
- Expression functionally related genes co-ordinately regulated

Question 5

“Split genes” in eukaryotic cells

Answer 5

Coding info in eukaryotic cells typically discontinuous in chromosomal DNA
Exons and introns, In need to be removed to bring coding sequence together
mRNA product typically smaller than original coding transcript
Prokaryotic mRNA coding transcripts have no introns so can be directly transcribed into proteins

Question 6

Intron-exon boundary sequences are conserved

Answer 6

In and Ex sequences distinguished through recognition splice site sequences (SSS)
5’ SSS Gu and 3’ SSS AG highly conserved
In also contain “branchpoint” A
Has consensus splicing signals
Conserved nucleotides are in bold

Question 7

Splicing mediated by spliceosome complexes

Answer 7

Pre-mRNA splicing carried out by spliceosome, large complex containing RNA and protein RNP - ribonuclear complex
Spliceosome very large RNP complex, larger than ribosomes
Active spliceosomes assembled and disassembled from smaller RNA/protein complexes called snurps (small nuclear RNPs)
This disassembly and reassembly makes it harder to see under microscopes

Question 8

The catalytic mechanism of splicing

Answer 8

Splicing involves 2 transesterification reactions:
1. 2’ hydroxyl group of branchpoint A attacks 3’ phosphate of 5’ exon
5’-2’ phosphodiester bond fives looped lariat
2. Generated 3’ hydroxyl group attacks 5’ phosphate of 3’ exon, releasing intron lariat
Therefore intron spliced from mRNA leaving exons
Intron lariat normally degraded by cell as not useful

Question 9

Catalytic RNA - ribozymes

Answer 9

Enzymes with RNA catalytic subunits
Nuclear pre-mRNA splicing thought to evolve from more structurally restricted, self-splicing introns
Spliceosomes thought evolve from self-splicing RNA (splicing in vitro)

Question 10

The RNA profile of Eukaryotic cells

Answer 10

Cellular RNA consist of:
- mRNA (~5%)
- rRNA (~75%)
- tRNA (~10%)
- small stable RNAs
mRNA not visible on gel as many different mRNAs exist in different sizes and generally unstable, also have v low abundance and only consist in cell for short length time when needed