L4 RNA Makes Protein Flashcards
What is a 5’ UTR and a 3’ UTR?
Refers to either of the two sections either side of the coding sequence (the open reading frame) in an mRNA
5’ UTR is the leader sequence
3’ URT is the trailer sequence
What is an open reading frame?
The coding sequence of mRNA (a reading frame that contains no stop codons)
Wobble base
When the third amino acid is irrelevant
Eg. Alanine can be GCA GCC GCG GCU
How is the ORF translated? And why is it like this?
I’m sets of three bases (codons)
Because it gives 64 possibilities of codes which is more than enough for 20 different amino acids to be coded for
What is the start codon
AUG
Methionine
Found as the first codon of the ORF
What are the stop codons ?
UAA
UAG
UGA
Don’t encode for any amino acids
Found as the last codon of the ORF
Give an example of something that uses a slightly different codon table
Mitochondria (use a slightly different codon table than in the cytoplasm)
How many Reading frames does each RNA have?
Three
However the start codon only occurs in one of the frames and so it determines the start of the correct ORF
Where is the start codon found in prokaryotes?
After the Shine-Dalgarno sequence
Which start codon determines the start of the frame in eukaryotes?
The first start codon found from the 5’ end of the mRNA
What is the sequence of the Shine-Dalgarno box?
5’ -AGGAGG- 3’
How does polycistronic mRNA work?
The shine dalgarno box sequence can occur multiple times in an mRNA sequence in prokaryotes.
This means in a single mRNA sequence there can be multiple proteins encoded (because multiple shine dalgarno sequences mean multiple sections of start codons)
What type of mRNA do prokaryotes have?
Polycistronic mRNA
Where multiple proteins are encoded for by a single mRNA
What type of mRNA do eukaryotes have?
+ what 2 otehr things does it have
Monocistronic RNA
(A single protein is encoded by the mRNA sequence)
5’ cap
Poly A tail
How can we locate the right Reading frame (cos there are 3 potential ones)
3/64 are stop codons
On average there are 16/1000 stop codons
ORF shouldn’t contain any stop codons
So we can estimate the correct frame that’s being translated because it’s the one where there are no stop codons in a section of about 1000 nucleotides
4 types of mutation
Silent
Missense
Nonsense
Frameshift
Silent mutation
Usually occurs in the last AA
UAU —> UAC (still encodes for tyrosine)
Missense mutation
UAU —> UCU (serine I stead of tyrosine) changed AA
Nonsense mutation
Introduces a stop codon
Eg. Tyrosine AUU can easily become AUG
Results in premature translation
Frameshift mutations
Caused by insertion or deletion of nucleotides (but if three nucleotides added/deleted frame stays the same)
Role of tRNA
Decodes the mRNA sequence into a protein sequence
Pairs anticodons and codons
Brings in charged AA
tRNA structure
At 3’ end there is a CCA where the amino acid becomes linked to this last A base Anticodon loop (middle loop) binds to the codon
What’s special about tRNAs recognising codons ?
In a cell there are about 30-40 tRNAs but as we know there are 64 codons
So one tRNA can recognise multiple different codons
What can uracil be chemically modified to?
Pseudouracil
Thymidine
Dihhdroudine
What can adenine be chemically modified to?
Inosine
Can bond uracil and cytosine both still pyramidine bases
What can guanine be chemically modified to?
Methylguanine
Explain how one tRNA can recognise several different codons ?
tRNA has a wobble base which is chemically modified after the tRNA is made
This changes the base into another base (other than the 4 main ones) which has the ability to bind to multiple bases, for example, uracil AND cytosine. (This is the case with inosine)
What shape does tRNA fold into?
L shape
Activated AA at 3’ end of L shape
Anticodon at other end
Explains tRNA biogenesis (three things)
tRNAs are spliced our form the precursor tRNA using ribozymes:
- RNAse P (splices at the 5’ end)
- RNAse D (splices at the 3’ end)
CCA is added to 3’ end by nucleotidyl transferase
Base modifications are guided by small nucleolar RNAs (snoRNAs) - RNA modifying RNA example of RNA word relic
Only after all of these does the tRNA have a charged amino acid added to it
How is tRNA charged?
Aminoacyl-tRNA-synthetase
Links the amino acid to the adenine base in CCA of the tRNA
One synthetase for each AA (20). This means that a synthetase must be able to recognise different codons which code for the same amino acid.
Nowadays: tRNA is charged with an AA and this is done by a protein
Before: good evidence it could have been down by RNA before. Flexizyme ribozyme discovered which could charge tRNA with AA
?
tRNA bound to glutamyl-tRNA-synthetase
One end of the synthetase enzyme recognises the anticodon on the tRNA (this synthetase is specific to the anticodon so it can bring the correct AA)
At 3’ end of tRNA is where the amino acid is linked to the 3’ OH of the tRNA
What’s the first step of the two step coupling of an amino acid to a tRNA?
Adenylation (activation) of the amino acid
- carboxyl group of AA reacts with phosphate in ATP so that pyrophosphate (two phosphates) is released and an ATP molecule with only one phosphate is now attached to the AA (see diagram in slide)
*example of an RNA based molecule (ATP) activating a small molecule (AA) - example of RNA work relic
What’s the second step of the two step coupling of an amino acid to a tRNA?
Transfer of the activated amino acid to the 3’ OH of the tRNA
(Same enzyme catalyses this)
What happens is incorrect AA is bound to tRNA?
If error found, enzymes can hydrolyse bond between AA and tRNA so no tRNA with the wrong AA is produced
Structure of ribosome
Made of RNA and protein Small and large subunit 80% of RNA in a cell is rRNA Prokaryotes 20AA/sec Eukaryotes 8AA/sec
Where does peptide synthesis occur
Large subunit of ribosome
Large ribosomal subunit
Oldest part of ribosome (from time when RNA was making peptides but not proteins)
Need evolution of small subunit to guide process and provide template for protein synthesis
Ribosome facts
30 nanometers
Polyribosome
Multiple ribosomes present on a single mRNA
S unit
Svedburg unit
Prokaryote ribosome
Large subunit - 50S
Small - 30S
Eukaryotic ribosome
Large subunit - 60S
Small - 40S
A site
Amino acyl site
Brings in tRNA with charged AA
P site
Peptidyl site
Contains tRNA that carries the peptide
E site
Exit site
tRNA that is in the exits site, it’s uncharged because the amino acid has been removed (added to the peptide) and this tRNA is about to leave
Where does anticodon-codon pairing occur?
Small sub unit near mRNA
Where does protein synthesis occur?
In the large sub unit at the peptidyl transfer centre
What are the three steps of translation
Initiation
Elongation
Termination
Initiation
1) small ribosomal subunit recognises and binds to the shine dalgarno sequence in the mRNA of prokaryotes (mRNA - rRNA interaction)
2) tRNA molecule charged with AA methionine binds with the start codon AUG that come after the SD sequence
Note: in prokaryotes the first methionine coded for because of the start codon has a blocked N terminus
3) large subunit binds to the complex of the tRNA with the charge amino acid (binds with the charge tRNA in the P site)
Elongation
1) tRNA bringing in a new AA binds to A site which puts it in close provoking to the already present peptide
2) peptide bond formed
3) translocation: ribosome shifts one codon in 3’ direction so the empty uncharged tRNA is now in the E site and ready to exit
4) empty tRNA exits E site
Peptide bond formation
tRNA carrying amino acid, amino group of this AA reacts with the carboxyl group of the already present peptide in the P site.
How does RNA catalyse peptide bond formation?
See slides
Base in ribosomal RNA that removes a proton from a nitrogen on the activated amino acid and creates a hydroxyl group !???????????
X
X
Termination
Occurs at stop codon
1) Release factor binds to stop codon
2) peptidyl-tRNA bond is hydrolyse so the final peptide product is released
3) both subunits, the tRNA and the RF dissociate from mRNA
What RNA world relics does translation involve?
mRNAs tRNAs and snoRNAs rRNAs RNAse P Adenylate-aa (activation of an AA through an adenylate)
Proteins involved
tRNA synthétases RNAse D Initiation factors Elongation factors Release factors
Why did proteins not takeover translation completely?
RNA is a relic from the past, probably irreplaceable part of current machinery, example of a living fossil that life has been built on
