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
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
