Term 2 Lecture 2: Translation: Degeneracy, Surveillance And Breaking The Code Flashcards
Degeneracy
More than one triplet code codes for the same as. All codons specify one AA and 64 codon options make 20 types of AA hence there is degeneracy in the code.
There are more than 20 tRNAs but not 64 as some match more than one codon arrangement The reason for this is because ‘wobble’ pairing at the 3rd base is relaxed.
The 3rd position in an Aminoacyl tRNA unit can tolerate a mismatch aka a “wobble” position. So for example tRNA^Ala1 has 2 alanine codons that it binds to GCC and GCU whilst tRNA^Ala2 has 2 other alanine codons it binds to GCA and GCG.
Quality control mechanisms
Errors in mRNA can lead to faulty proteins
‘mRNA surveillance’ helps stop cells from making useless or toxic versions of proteins.
2 mechanisms are used:
1) nonsense mediated delay (NMD) which occurs in eukaryotes and eliminates mRNAs with premature stop codons (not covered at this level)
2) tmRNA (transfer mRNA) fusion that occurs in prokaryotes to avoid accumulation of deleterious proteins
The problem with non-stop mRNA and how tmRNA fixes it
If there is no stop codon on an mRNA due to a mistake or break in the mRNA the ribosome is unable to release this mRNA once bound.
1) the ribosome stalls when it reaches the end of an mRNA lacking a stop codon - waiting for a release factor that will not come
2) tmRNA charged with an Ala molecule binds to the A site of the ribosome and alanine is added to the polypeptide chain.
3) the tmRNA consists of a code for (usually) 9 more AA that are then added to the existing polypeptide chain in the stalled ribosome
4) the stop codon from the tmRNA moves into the A site and the polypeptide chain and mRNA is released
5) the 10 aa ‘tag’ (Ala+9 more aas) labels the produced protein as faulty targeting it for degredation by the cell - preventing it doing anything
tmRNA summary
-produces labelled aberrant protein that the cell can recognize and target for degredation.
- not clear exactly how it is recognised
- some evidence that the system also allows detection and destruction of aberrant mRNA too
Why does it have to be a triplet code?
- needs to encode 20 aa so needs at least 20 combinations
- doublet base code would give 4² combinations (16)
- triplet base code gives 4³ so 64 combinations
- quadruplet base code 4⁴ would give too many
Using Occam’s razor theorem
Theorem: Usually the explanation that requires the fewest assumptions is correct.
E.g. on hearing the sound of hooves in England we would assume a horse is approaching and not a zebra
Therefore we assumed the triplet base code - there is no need for more than 20 aas - 4² is too few and 4⁴ is more than is necessary therefore we assume there is a triplet code - no need for more complication.
Why does it have to be a triplet code?
Crick et al. 1961 looked at FCO mutant of bacteriophage T4 and made further mutation to see the effect on the phenotype (visual appearance)
FCO mutant is a base insertion causing frameshift that affects the phenotype.
They continued to make a series of mutations to the FCO mutant to convert it back to wild type ( with the assumption that mutation always added or removed one base)
Suppression of FCO removes a baze countering FCK base introduction mutation.
Addition of 2 bases makes a mutant
Addition of 3 bases returned it to wild type as did removing 3 bases - showing that bases are in coding groups of 3
Marshall W. Nirenberg - creating the code
American biochemist and geneticist at NIH Maryland in the early 1960’s
Nirenberg decided he could design a system to test which codons relate to which AA with his post doc assistant Johann Mattaei.
They published their first code cracking paper in 1961 and completed all other AA codes earning a Nobel prize in 1968
Severo Ochoa
A competitor to Nirenberg, a senior scientist born in Spain working at the NY medical school.
Won the physiology/medicine Nobel prize in 1959.
Studied synthesis of RNA and DNA
Ochoa was an established scientist with a large team.
He discovered polynucleotide phosphorylase enzyme existing in nature and found that it can be used in labs to construct long RNA molecules
Nirenbergs first experiment
Used polynucleotide phosphorylase to make his own artificial RNA molecules to see how cells respond and what proteins they make.
He used a cell free extract - created by killing a cell and extracting its machinery to use it in vitro (usually in a test tube)
Process
1)He used E. coli from a culture, crushed and filtered it to form a cell free extract containing everything required to synthesise proteins - ribosomes, tRNA etc.
2)First he left the extract long enough for the mRNA to fully degrade (it’s fairly unstable) until no RNA remained in the system.
3)Then the cell free extract was treated with DNAse to kill DNA preventing it’s transcription to RNA.
4)He used polynucleotide phosphorylase to make homolymers e.g. AAAAAA… Or UUUUU…
5)He labelled each test tube contents with an AA specific radiolabel
He then added these long homopolymer chains to the test tubes of extract and left them to translate.
6)As there is no other RNA in the extract only what he’s added is translated.
If that particular codon e.g. uuu. Does for the specific radioactive aa then radioactive protein can be detected - if it specifies another protein it won’t be detectable radioactively.
Nirenbergs first experiment breakthrough and the problem
UUU coded for Phe
poly U homopolymer was added to the cell free protein synthesis system and the protein produced was a chain entirely made of Phe detected by its radiolabel.
The problem
Nirenberg and his team tested all 4 homopolymer triplet GGG,AAA,UUU and CCC. After that they tried feeding the system chains of mixed bases but there was no way to decipher which triplet combinations were coding the resulting proteins.
E.g. An A and C mixed polynucleotide chain could have 8 variations : AAA, AAC,ACC,ACA,CAA,CCA,CAC or CCC and they couldn’t know which combination was being made as they had no control of base order.
Even if the proportions of two bases was controlled there were many possible arrangements.
Har Gobind Khorana 1963-66
Born in Rajpur Punjab he studied at Liverpool for a PHD and then at the university of British Columbia and Wisconsin USA
Niche in chemical synthesis of nucleotides
Before then noone could specify which nucleotide they wanted to make and could only get an enzyme to string together what was there.
Khoranas method allowed RNA molecules to be designed and tested to see what the system makes from them
He achieved a direct synthesis of bases in a particular order (specified by the scientist and not by nature)
Khoranas experiment
He made simple RNA polymers to begin with - just patterns of 2 alternating bases e.g. GuGUGUGU…
Found to code Val,Cys,Val,Cys…
The resulting proteins had two alternating aas. From this he knew there were only two codon possibilities GUG and UGU that coded 2 different AA.
Further experiments allowed him to work out which codon coded each AA.
It was still not possible to work out every codon and there were some anomalies - the method needed further adaptation.
Working out the other codon combinations
Nirenberg and Leader discovered that trinucleotides (codons) promote the binding of Aminoacyl tRNA to ribosomes.
Using Khoranas chemical system they synthesised all 64 triplet combinations
By supplying one triplet codon with all bases and adding it to 20 test tubes each containing cell free medium radio labelled to detect 1 of the 20 amino acids it was possible to see which codons synthesised which aas.
Process
1) synthetic mRNA of one codon is synthesised
2) codon is added to a mix of ribosomes and Aminoacyl tRNAs
3) the ribosome bound to the mRNA and the Aminoacyl tRNAs specified by the mRNA
4) filter solution - mix is passed through a nitrocellulose filter. tRNAs paired with ribosome bound mRNA stick to the filter and unbound tRNAs pass through
5) filter is assayed to determine which AA was bound
1968 Nobel prize in physiology of medicine was shared by Nirenberg, Khorana and Robert Holley (who discovered tRNA structure). They shared the prize for their interpretation of the genetic code and it’s functions in protein synthesis
