Translation Flashcards
Translation is described as endergonic
Meaning what
driven by high-energy phosphoanhydride bond cleavage
Why are 3 bases used to form a codon rather than two
there are 4 bases (A,U,C and G) and there are 20 naturally occuring amino acids
Hence a doublet of 2 bases per codon would be insufficient: 4^2 = 16 doublets
A triplet code (3 bases per codon is sufficient: 4^3 = 64 different triplets of bases
If there are 20 naturally occuring amino acids and 4^3=63 different triplets of bases, what does this mean
A triplet code allows amino acids to be specificed by ≥ 1 codon
This means DNA is degenerative
Apart from being degenerative what is another feature of DNA
Genetic code is non-overlapping
Meaning each triplet is read seperately without any repeats
This means DNA has to be longer, however there is less risk of major issues from mutations
What did Francis Crick, Sydney Brenner discover in their experiment together and what two things did it show about the nature of DNA
Utilising bacteriophage (bacteria eating virus) T4, they discovered that a deletion of a nucleotide could abolish gene function
A 2nd mutation (an insertion close to it) could restore the gene function
‘the mutations are suppressors of another’
Thus genetic code is read sequentially from a fixed point ‘reading frame’
Further work showed that 2 closely space deltions/insetions could not resotre gene function but 3 could - giving evidence for the triplet
Hence, insertions and deletions are known as what type of mutations
Frameshift mutations
True of False:
mRNA does not directly recognise amino acids
True
mRNA binds to molecules of tRNA (carrying amino acids)
each tRNA contains an ‘anticodon’ which is complimentary to the mRNA codon
Any nucleic acid may have 3 ‘active’ reading frames. What does this mean
Therefore 1 polynucleotide could encode up to 3 polypeptides
In the 1960s sequencing wasnt available, meaning they could link parts of the mRNA sequency to part on the protein
What experiment done by Nirenberg and Matthaei was used to overcome this
Instead they used an enzyme called polynucleotide phosphorylase from a bacteria
If u react it with nucleotides it will join the nucleotides together - hence creating artifical bits of mRNA. This then could then go through protein synthesis and find out what proteins are formed
In the 60s, once artifical bits of RNA where created, how did scientist convert this into protein
E.coil cells were broken open and centrifuged to remove cell wall etc (hence DNA, mRNA, ribosomes and enzymes where left) - left with a cell free system
They have to stop the cell free system from producing any of its own proteins using DNase to remove DNA (halt synthesis)
The synthetic RNA was added (and tRNAs which where radiolabelled)
What was the outcome of the 1961 experiment by Nirenberg and Matthaei
It hows that Phenylalanine which was coded by the codon UUU
Lysine was coded by AAA
Proline was coded by CCC
How else could an experiment be carried out to work out how mRNA codons relate to the proteins formed
This experiment in particular allowed 50 codons and their amino acids to be worked out using pre-made mRNA
The ribosomes were bound to a nitrocellulose filter and give them the mRNA you’ve created
You then provide the entire mix of tRNA with radiolabelled amino acids bound to them, which are washed through the nitrocellulose filter
Some of them will start sticking to the mRNA present there in
Then you can analyse what is being produced
What was the work of H.Gobind Khorana
Chemically synthesised polynucleotides with repeating sequences (the UC sequence)
This was fed into the cell free ribsome system which allowed the creation of different peptides (depending on where the reading frame begins
This allowed the last few codons to be decifered
Leucine has 6 different codons which can be used to code for this amino acid. What is the benefit of this
That if there was a mutation, it is less likely to mean that a different amino acid would be codes for, hence there would be a limited impact on the protein formed
Coding codons for the same amino acid are know as synonomous (they differ only in their third nucleotide)
What do you call a codon which does not code for an amino acid
A non-sense codon
They are likely to be found at the end of a mRNA sequence
These are UAG, UAA, UGA (stop codons)
AUG and GUG are start codons (but also will code for Met and Val at interal positions)
Changes in the 1st position cause
Specify similar amino acids
2nd position pyrimidies encode most …
… hydrophobic amino acids
2nd position purine encode …
… mostly polar amino acids
True or False
Genetic code is universal
False
Not all organisms actually utilise the code
Certain mitocondria, were showing varieties of the genetic code
UGA translates tryptophan rather than stop
There is also alternate genetic code in ciliated protozoa as well which branched off in early eukaryotic evolution
most tRNA have a similar structure
What are some key features
54-100 nucleotides in length
Arranged in a cloverleaf structure
- There is a 5’-terminal phosphate group (3’ hydroxy group is where the amino acid will bind) known as the acceptor stem
- You do not have your usual Watsona dn Crick bases e.g. G with a Me group attached to it
- The ‘D-arm’ ends in a loop containing dihydrouridine
- The anticodon arm - loop containing the anticodon
The bases on tRNA are not the usual Watson and Crick bases
Why have we evolved this way?
We don’t think they’re absolutely necessary for mantaining the integrity of tRNA
But they are good at supporting the amino acid attahment to the acceptor stem
And potentially strengthen codon-anticodon interactions
In 2D tRNA is a cloverleaf shape, however what is its shape like in 3D
tRNA has a complex 3D structure which is an L-shape
Where most of the bases are hidden from the surrounding solution, except for the ones at the 3’ end and also the anticodon loop
Why has tRNA evolved to have this 3D tertiary structure
the molecule is particularly narrow
tRNA work in close proximity to another at the ribsosome, it has evolved to have thin structures like this to bind in close proimity on adjacent codons
Translation must also have a level of accuracy
How is this done
Recognising the correct codons
making sure tRNA has bound to the right amino acid
The enzyme that does this is aminoacly-tRNA synthase which attaches amino acids
Aminoacylation occurs in 2 steps
1) activate the amino acid by sticking an adenosine monophosphate on one end,
2) which interacts with the tRNA to form aminoacyl-tRNA
This process is driven due to pyrophospahte being produced which is an energy rich bond (pulling reaction in favour of products)
There is also more than one tRNA that will carry the same amino acid as another
What is the name for this
How does this occur
Isoaccepting tRNA
Must all be recognised by aminoacyl-tRNA synthase as it loads the amino acids onto the tRNA
It does this by contacting the tRNA in the acceptor stem and the anticodon loop
How are mistakes in the amino acid sequence prevented during the translation process
Proofreading
Isoleucines aminoacyl-tRNA synthase
This will stick on 40,000 isoleucines onto its correct tRNA for every accidental incorportation of valine
Valine only differs by a sinlge methyl group
Protein synthesis requires the proper tRNA being selected for via codon-anticodon interactions
But, each of the 61 codons are not read by different tRNA
This can be caused by what
Many tRNA bind to 2 or 3 codons - this is known as wobble
Non-Watson-crick base pairing can occur at the 3rd codon-anticodon position
e.g. Guanine with a methy group attached meaning not only can it complementary base pair with cytosine (normal), it can also with uracil
What feature of genetic code does wobble account for
Degeneracy
First 2 codon-anticodon parings = Watson-Crick base pairing
Wobble at 3rd position
Organisms will preferentially have certain codons encoded for in their genome rather than other
What can this be used for
A timing impliment during protein synthesis
For example if you wanted to create a protein fairly rarely, you may put to a less frequently used codon in the middle of the RNA, meaning it would take longer for the correct tRNA to meet it and hence produce your protein
In 1955, Paul Zamencnik identified where as the site of protein synthesis
Ribosomes
Ribosomes are large
What is the difference in size between Bacteria and Eukaryotes of their ribosomes
Bacteria = 2.5 x 10^6 daltons
Eukaryotes = 3.9-4.5 x 10^6 daltons
So slighly bigger in eukaryotes
Ribosomes are complex macromolecules, made up of which two biological molecules
RNA molecule
Multiple proteins
What are the functions of ribosomes
- Binds mRNA, so that codons can be read
- Include binding sites for the tRNA molecules
- Mediate interactiond of non-ribosomal protein factors:
polypeptide chain initiation, elongation and termination - Catalyse peptide bond formation (only cell which can do this)
- To be capable of movement along the mRNA
The prokaryotic ribosome is known as
70s ribosome
What are the ribosomes made up of in eukaryotic and prokaryotic cells
2 subunits
A small and a large one
In prokaryotic ribosomes (70s), the small and large subunits are made up of how much amino acid and different proteins
Small (30s) = 16s rRNA and 21 proteins
Large (50s) = 5s and 23s rRNA and 31 different proteins
In E.coil there are up to 20,000 ribosomes which accounts for 80% of the RNA and 10% of cellular proteins
This shows what
Significant part of the organelle used in protein sythesis
Ribosomal protein are located where
on subunit back and sides and not near the tRNA and mRNA binding sites
This has a stabilising function
Ribosomes also have a site of what
What is significant about its location
Catalysis - called the ribozyme (catalytic part of RNA)
which is is very far away from the nearest protein and RNA actual does the job of catalysisng the peptide bond formation
An example of ribozyme IRL
Introns in heterogenous RNA will catalyse their own removal
A ribosome has how many t-RNA binding sites
3 t-RNA binding sites
1. The ‘A-site’ (aminoacyl site) - accomodate the incoming aminoacyl-tRNA (tRNA bound to an amino acid)
2. The ‘P-site’ (peptidyl site) - accommodates the tRNa attached to growing peptide chain
3. The ‘E-site’ (exit site) - where the empty tRNA which has lost its amino acid, that is leaving
The t-RNA is very thin in its 3D structure.
How does this relate to its 3 binding sites
such that a peptide bond can form between the amino acid on the A sight, and the elongating protein chain on the P site
Allows a close fit of tRNAs together, on the ribosome
Eukaryotic ribosomes are 40% bigger than bacterial versions
What is the name for the Eukaryotic ribosomes, and what is the ratio or protein to RNA in their small and large subunit
80s
Small subunit (40s): 33 polypeptides and 18s rRNA
Large subunit (60s): 49 polypeptides, 28S, 5.8s and 5S rRNA
Apart from the ribosome, the stages of translation (initation, elongation and termination) require factors for peptide synthesis
They can be
Catalytic or stabilise
What are the two energy sources required for translation to occur
ATP and GTP
(more than two GTP are required - one to bind to aminoacyl-tRNA to A site and 1 for translocation)
This is due to it being a polyerimerisation reaction
How does the tRNA move through the binding sites on the ribosome during chain elongation
Growing polypeptide is transferred from ‘P-site’ on the peptidyl-tRNA to the incoming aminoacyl-tRNA in the ‘A-site’
The new peptidyl-tRNA is transferred from the A to the P-site
The uncharged t-RNA moves to the E site
Prokaryotic mRNA are polycistronic, meaning what
Eukarytic mRNA is monocistrinic, meaning what
What is a polysome complex
Prokaryotic: can have more than more coding region
Each coding regions has its own initiation and termination codons
Eukaryotic: mRNA have just 1 coding region
But because of the length of mRNA, more than one ribosome can translate a messgage (forming a ‘polysome’ complex)
How does the polycistronic nature of prokaryotic cells mean prokaryotes can produce proteins very quickly
As soon as prokaryotes starts to produce mRNA from the gene during transcription, not only one, but multiple ribosomes can bind to it.
This is in addition to having no introns
What is meant by the initiation phase
Everything that’s happening before the first peptide bond formation
What is require for the initiation phase to occur
2 ribosomal subunits
mRNA to be translated
Aminoacyl-tRNA specificed by the 1st codon
GTP
Initiation factors (in prokaryotes IF1,2 and 3)
What are the steps involved in initation
The ribosome has to be constructed around the RNA in a way to allow translation to occur - want mRNA lined up so that the start codon can be regonised in the correct place of the ribosome
In Eukaryotes: its done by using the cap structure at the end of the RNA to work out how to align itself
Prokarytoes: have multiple proteins encode for by the same RNA. So this relies on the ‘shine-Dalgarno sequence’
What is the Shine Dalgarno Sequence
Is a purine rich section of the mRNA, which will complimentary base pair with a pyrmidine rich section of the 16S rRNA
This means the start codon is in the right position for translation to begin
True of False
tRNA always enter the A site first
False
The initiating AUG codon is recognised by special tRNA which goes straight to the small subunit P-site
Facilitated by eIF-2-GTP in eukaryotes (IF-2-GTP in prokaryotes)
The large subunit then joins the complex
What is the main role of the 23S ribsome part in prokaryotes
It carries out the peptide bond formation
What do the Elongation factors do
Elongation factors EF-Tu-GTP brings the appropriate charged tRNA to the codon in the empty A-site
GTP on EF-Tu is hydrolysed
What is translocation
Where the ribosome advances 3 nucleotides towards the mRNA 3’ end
What group are amino acids added to on the growing polypeptide
The carboxyl end
What happens to the onced charged tRNA in the P-site, once the ribosome moves along the mRNA
Now moves to the E-site
What was in the A site now takes its place
How does the termination of translation occur
A non-sense codon will be reached, where no tRNA will be bound
There will be release factors which recognise the empty codon in the A site. GTP will be hydrolysed
The ribosome and it’s elongating polypeptide will actually dissociate from one another
How is the protein relased from the tRNA
Release factors binding causes hydrolysis of the bond linking peptide to tRNA in the P-site
This protein will then undergo post-translation processing
Most antibiotics we use to combat bacteria, will affect translation
Streptomycin is used to treat Terberculosis, How?
You will see a different affect on if a low or high dose is given
Low: leads to the mRNA being misread. Pyrimidines may be mistaken in 1st & 2nd codon position - leading to amino acids being encorporated with different physiochemical properties
More likely high dose: prevents chain initiation - the ribosome cannot form around the mRNA - causing cell death
How does Tetracycline (antibiotic) work
It binds to the small prokaryotic subunit of the ribosome, which prevent entry of the aminoacyl-tRNA into the A-site - stopping translation.
But it doesn’t stop the elongating factor from using energy from GTP to deliver the amino acid (energy drain on the bateria)
How does Chloramphenicol (antibiotic) work
Does affect eukaryotic translation however
It binds near the A-site and stops the peptide bond formation
Once translation is terminated, the polypeptide formed will be released from the complex. It then will need help to fold in post-translational modification
What molecule allows this
Ribosome associated Chaperones will help a protein to fold
What is cotranslational modification
If the polypeptide is still attached to the ribosome
What is posttranslational modification
After synthesis
Many polypeptides are enzymes
How do these enzymes become activated
Specialised endoproteases activate the molecule
In their unactive forms they are known as zymogens
What is another way proteins can be activated
By covalent attachment of chemical groups
Phosphorylation: occurs on the hydroxyl group of serine, threonine and tyrosie resides, which will cause a change in charges, hence structure. Catalysed by kinases
Another way to modify the protein is to glycosylate them
What does this involve
Sticking sugars on the protein
Many proteins are designed to be secreted or part of the cell membrane have carbohydrate chains attached
Or act as cell recognition particles
Will happen in the golgi
When proteins reach the end of their life span, they also can be modified - tagged for distruction
How does this happen
Ubiquitin can be added to them
This can attract the activity of the proteosome, which will then recycle them
