RNA Makes Protein Flashcards
Why do we need proteins?
They are better catalysts
What two sections does mRNA contain?
- ORFs (open reading frames)
- UTRs (untranslated regions)
Describe ORFs
Contain 3 nucleotide codons, each for one aa
What is the wobble base?
The third base, that is often irrelevant for coding
Start codon
AUG
Stop codon
- UAA
- UAG
- UGA
How is translation initiation achieved in prokaryotes?
- polycistronic mRNA
- ribosome binds to Shine-Dalgarno box
What is polycistronic mRNA?
Encodes multiple proteins
Shine-Dalgarno box
5’-AGGAGG-3’
How is translation initiation achieved in eukaryotes?
- monocistronic mRNA with a 5’ cap and poly-A tail
- ribosome binds to 5’ cap and scans for first AUG
What predicts which frame is translated in mRNA?
- stop codon distribution
- statistically, averages 3/64 codons, 3/192nts = 15.6 per 1000nt
Silent mutations
- do not change aa sequence
- ~1/3
Missense mutations
- cause aa substitutions
- ~2/3
Nonsense mutations
- cause premature termination
- <5%
Frameshifts mutations
- Caused by indels
- often truncate proteins
What is the function of tRNA?
To decode the mRNA sequence into protein sequence
What is the function of rRNA?
the main component of the ribosome - the translation machinery
tRNA facts
- 15% of all cell RNA
- 78-91nts
- 31-41 per cell
- 3’ end carries activated aa
- clover shaped secondary structure
- one tRNA can recognise several codon
- ~20% is modified RNA bases
What is the 3’ end sequence of tRNA
CCA
What is the middle loop in tRNA?
The anticodon loop
List the modified RNA bases
- Dihydrouridine
- Inosine
- Ribothymidine
- Pseudouridine
Describe Pseudouracil
Has nitrogen replacing carbon 5
Describe inosine
O = relaxes NH2
Describe dihydrouridine
Loss of carbon chain
Describe methyl-guanine
Methylation of amine group
Structure of tRNA
- folds in L shape
- D- and TψCG-loops interact
- anticodon on one end of L
- activated aa on the other end of the L
- flexibility (twisting) in variable loop
- acceptor stem
tRNA biogenesis
- tRNA precursors processed at 5’ end by ribozyme RNAse P
- tRNA precursors processed at 3’ end by enzyme RNAse D
- tRNA nucleotidyl transferase adds 3’ CCA
- base modification of snoRNAs
- splicing (for some)
What are tRNA precursors?
Often tandem arrays with different tRNAs
snoRNAs
small nucleolar RNAs
Flexizyme
Artificial 42nt ribozyme that can charge tRNA with aa
aminoacyl-tRNA synthetase
aaRS
charging tRNA with activated aa is
a highly selective coupling reaction
How is an amino acid activated
- aminoacyl-tRNA synthetase catalyses ATP + amino acid -> AMP
How many aaRS for each aa?
1
How many aaRS are there?
- 20
- some can recognise multiple tRNAs (because there are 31-41 tRNAs)
Charging tRNA with activated aa mechanism
- coupling of aa to 3’OH 
- specific anticodon recognition creates an RNA-protein complex
Describe the coupling of the aa to 3’OH of tRNA
- Adenylation of amino acid with ATP (pyrophosphate byproduct)
- Amino acid transfer to tRNA 3’OH, releasing AMP
- Hydrolytic editing to remove misincorporations
Describe the error rate of hydrolytic editing
~10-6
- due to highly selective coupling
Speed of translation in prokaryotes
17-21 aa/sec
Speed of translation in eukaryotes
- 6-9 aa / sec
- 1-2 proteins per minute
Describe the ribosomal large subunit
- contains peptidyl transferase
- possibly oldest part of ribosome
- RNA -> peptides possible
Describe the ribosomal small subunit
- contains mRNA guide
- probably evolved later
- allows RNA -> proteins (true translation)
How big are ribosomes?
- big!
- 30nm
- can be seen under TEM
Polyribosomes
Multiple ribosomes per mRNA (think beads on a string)
What is the Svedberg unit?
- S
- non-metric unit for sedimentation coefficient
What is Da?
- Dalton
- atomic mass
- H = 1Da
Where is tRNA in the ribosome?
- A- site (amino acyl)
- P- site (peptidyl)
- E-site (Exit)
What are the 3 steps of translation:
1) initiation at start codon
2) polypeptide elongation
3) termination at stop codon
Describe translation initiation in bacteria
- blocked N terminus
- small subunit binds to Shine-Dalgarno
- fMet-tRNA binds to AUG start codon
- large subunit binds to fMet-tRNA in P-site
What is a blocked N-terminus?
N-formyl methionine is used as the first amino acid
What does translation initiation in bacteria involve at the small subunit?
- mRNA-rRNA interactions
- mRNA-tRNA interactions
Describe elongation
1) acyl-tRNA loaded into A-site
2) peptidyl transfer
3) translocation, peptide-tRNA in P-site
4) empty tRNA exits E-site
What happens during peptidyl transfer?
Peptide bond formation
What is translocation during translation?
ribosome shifts one codon in 3’ direction
Describe peptide bond formation
- amino group of incoming amino acid attacks C-terminal carbonyl of peptidyl-tRNA
- transfer of peptide into A-site
How does RNA catalyse peptide bond formation?
- rRNA adenine accepts proton from NH3+
- donates proton to hydrolyse tRNA peptide in P-site
The catalytic centre is
Adenosine
Describe termination
1) RF binds to stop codon
2) peptidyl-tRNA bond hydrolysed; product release
3) subunits, tRNA and RF dissociate from mRNA
RF
- release factor
- protein
In what direction is RNA translated?
5’ -> 3’
At what end is the untranslated leader?
5’
At what end is the terminator?
3’
Open reading frame is usually much longer than
1000 nucleotides
Where does codon-anticodon coupling happen?
In the small subunit
Where is the peptidyl transfer centre?
In the catalytic site, the large subunit
Where is 16s rRNA
small subunit
During termination, the bond between peptide and tRNA is
Hydrolysed
