The genetic code and transcription II Flashcards
in-vitro protein synthesis
Carrying out a biological reaction in a non-biological system
[ i.e. in a test tube]
- In order to undertake in vitro protein synthesis, we need a cell-free extract
- The cell extract is obtained by lysing the cells and then centrifuging out all the debris
- what remains is all necessary cellular machinery
- In order to track this synthesis, we need one or more radioactively labeled amino acids - which must be supplied by the amino acid mixture which is used.
HOWEVER:
The clever part of the experiment was in the synthetic mRNA sequences they used for the polypeptide synthesis
after finding out what the genetic code looks like, the next step was to decipher the code
Nirenberg & Matthaei took the first steps in characterizing important codons
They used:
- in vitro protein synthesis (components)
- Enzyme: Polynucleotide phosphorylase
- Using this enzyme: they made synthetic mRNA’s which they could
use as templates for polypeptide synthesis in a cell-free system
> No DNA template
- Polynucleotide phosphorylase actually degrades RNA in living
cells - broken down- releases ribonucleoside diphosphates
HOWEVER, this reaction can be reversed (forced) (towards synthesis) - Given that the enzyme is isolated and exposed to very high
concentrations of ribonucleoside diphosphates - It doesn’t need a DNA template
- The order of nucleotides which is added to the growing RNA in this forced rxn is random and depends entirely on the relative concentrations of the 4 nucleoside diphosphates that are present in the reaction
i.e. VERY IMPORTANT:
The probability of the insertion of any nucleotide is entirely proportional to the availability of that nucleotide relative to all the other nucleotides in the rxn mixture
To keep it simple - Nirenberg and Matthaei first synthesized RNA homopolymers:
- They only supplied one type of ribonucleotide - so that the entire polymer consisted of that base only
UUUUUUUUU – poly-phenylalanine
AAA = lysine; CCC = proline; GGG = ?
They then tested mRNA (AAA, CCC, GGG, UUU) in the in vitro protein synthesis system and looked at the amino acids which were incorporated into the proteins which were synthesized
- To detect which amino acids were incorporated, they repeated the experiment 20 times - each time making sure that all 20 amino acids were used - by using one differently labeled amino acid each time
- So that they could tell when the labeled amino acid was incorporated into the protein
When they used uracil (polyuric acid) in an experiment where they also added 14 carbon phenylalanine:
- They produced a phenylalanine polypeptide
[UUUUUUUUU – poly-phenylalanine]
- From this, they concluded that the codon, UUU, encoded for phenylalanine
Using similar experiments they found that:
AAA = lysine;
CCC = proline;
GGG = ? - probably because molecule folds back on itself
[Telomeres with the G quartets]
Could only find this because they used homopolymers - didn’t have other means at the time
Translation
Consists of various steps which can be divided into 3 basic processes:
Initiation, elongation and termination.
Components of Translation
- Large and small ribosomal subunits
- mRNA
- Charged tRNA’s
- Peptidyl transferase
- GTP & Mg++
- Initiation factors (IF1, 2, 3)
- Elongation factors (EF-Tu, EF-G)
RNA heteropolymers
(Nirenberg et al.)
- next, they used, mathematical predictions to help them assign
amino acids to codons containing more than one type of
ribonucleotide
BECAUSE:
The probability of the incorporation of a nucleotide is prop. to its relative concentration
They could mathematically predict frequency of each possible codon
Probability:
[no. of nucleotides in triplet]
(proportion)^
Mixed copolymers
Specific rNDP ratios in solution
E.g: (C:A = 5:1)
Go to slide 6 in The genetic code and transcription II PP!!!
Triplet Binding Assay
(Nirenberg & Leder, 1964)
- To help them assign specific sequences to the various amino acids
> The ribosomes (in vitro) binds to RNA triplet (length of codon) and attracts complementary sequence in charged tRNA
(e.g. UUU attracts tRNA-phe)
The tRNA contains the amino acid at one end and an anti-codon that is complementary to the codon on the other end.
By doing this, they could create a complex that would attract the right amino acid to the particular codon
The system they used was rather simple:
- The amino acid they wanted to test was radio-labeled with C14 and then combined with its cognate tRNA - creates a charged tRNA molecule
- Using the base compositions they already worked out for each codon- it was easy to decide which amino acid to test for which triplet
- The radioactively charged tRNA, RNA triplet, and ribosomes were then mixed together and placed on a nitrocellulose filter.
- The nitrocellulose filters are fine enough to retain the ribosomes but not the other molecules
- So if no radioactivity remained on the filter, then the tRNA containing the amino acid washed through and the wrong amino acid had been tested
HOWEVER:
- If the radioactivity remained on the filter, then the charged tRNA had bound to the codon that was in the ribosome
- This allowed them to assign specific codon sequences to specific amino acids
- Codons of known sequence could be synthesized to act as
mRNA
- 50/64 triplets assigned to specific amino acids
- Confirms that the code is 1. degenerate and 2. unambiguous
1. More than one codon for most of the amino acids
2. Each codon only attracted a single charged tRNA - thus coded
for only one amino acid
Repeating copolymers
(Khorana)
- An alternative method for deciphering the genetic code
- He synthesized longer RNA molecules consisting of short
sequences that were repeated many times
1. First he created di-, tri- and tetranucleotide sequences
2. and then he replicated them many times
3. And then finally he combined them enzymatically to form longer polynucleotides
Process of elimination
Stop codons
Listen to slide 9 of The genetic code and transcription II
The Coding Dictionary
- 61 of the 64 triplet codons = 20 amino acids
- The remaining 3 = termination signals - don’t specify amino acids
Met = initiation signal
Degenerate nature – Met and Trp only one codon each
Unambiguous, any codon can encode only one AA
“Ordered” degeneracy
Codons from a class of amino acids often share middle base
U/C - hydrophobic
G/C – hydrophilic
Buffers the severe impact of mutations on fidelity of translation
(impact on protein structure)
Crick’s “wobble” hypothesis
- Codons for the same amino acid often differ only in 3rd base
- (During translation) First two bases are most important in attracting anticodon of tRNA
- More flexible base-pairing at position 3 (wobble nucleotide)
> it is less spacially constraint than the other 2 in the ribosome
> Allows flexibility in base pairing - Allows a potential anti codon to pair with more than one codon
Economy measure
Initiation, termination and suppression
in vivo- protein synthesis is a highly coordinated and organized process
- Initiation of a protein only ever begins at an AUG (start) codon
In bacteria:
- the polypeptide chain grows from an inserted formyl methionine
(fmet)
- AUG is the only codon that can code for methionine
- and when this codon occurs internally in a bacterial mRNA -
standard methionine is inserted
- Once translation is completed, the formyl residue is removed from the starting methionine
> Although, sometimes the entire fmet molecule will be removed
In eukaryotes:
Standard met. (AUG) is the starting codon and can also be removed
- The three termination codons don’t encode any amino acids
- They act to terminate protein synthesis
- they are not recognized by any of the tRNA species
Thus, the translation process is terminated when they are encountered on an mRNA template
Nonsense mutation
If a mutation changes any of the other codons in an mRNA molecule into a stop codon, then protein synthesis will be terminated early
= only a partial polypeptide being synthesized
Initiation, termination and suppression continued
Phage MS2:
- is a bacteriophage that infects E.coli
- Has a very small genome with only 3.5 thousand ribonucleotides
> which encodes only 3 genes
This simple system allowed researchers to sequence the genes and their products
- When the nucleotide sequences were aligned with their encoded
proteins- they were found to be collinear
- This means that the linear sequence of nucleotides (in their codons) aligned exactly with the linear sequence of amino acids encoded by those codons in the protein
- They also noticed that the sequence began with AUG and terminated in a pair of stop codons
There is evidence that the code is commonly shared across viruses, pro and eukaryotes and archaea
Universal nature – exceptions in mitochondrial DNA
Exeptions to the Universal nature of codon dictionary
– exceptions in mitochondrial DNA
E.g:
UGA- usually encodes a stop codon- was found in the middle of some mitochondrial genes
other exceptions have been found in some bacteria and protozoans
- Its been noticed that generally the change involves only the
wobble nucleotide
- It has been suggested that this could represent an evolutionary shift towards reducing the number of tRNA’s that are needed in mitochondria
E.g: in human Mit. have 22 tRNA species (half the num. found in human cells)
