The in vitro approach has the following four methods:
1. Genetic analysis of deletion mutatants of a cistron,
2. Incorporation of amino acids by synthetic polyribonucleotides
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3. Binding of known triplets of aminoacyl tRNA.
4. Amino acid sequence generated by mRNA transcribed from synthetic tRNA of known nucleotides sequences
1. Genetic analysis:
Genetic analysis of deletion and insertion mutants (spontaneous as well as induced) in phage T4 was reported by Crick et al in 1961.
This deletion or insertion of a nucleotide in a DNA chain leads to a frame shift mutation after the point of deletion or insertion.
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As a result of this, the reading of the codon subsequent to the mutation gets altered. Obviously then the amino acid composition of the resultant protein would be different. As a result, the protein may be functionally inactive.
All the mutations tested by Crick et al produced a phenotype called mutant II. It was further observed by Crick et al that a series of three mutations was necessary to suppress the effects of a previous mutation.
In other words three suppressor mutations would nullify the effect of three frame shift mutations and bring back the original phenotype.
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This is a clear evidence to show that the altered codon reading would return to the normal state after three point mutations thus indicating that the codon must have three nucleotides.
The study also indicates that the code is comma less. If there were to be a punctuation mark between the two codes a deletion or insertion of a base would change the number of bases between two commas adjacent to the point of insertion or deletion.
2. Incorporation of amino acids by synthetic polyribonucleotides:
Synthetic polyribo nucleotides were used as mRNA by Nirenberg and Matthai to determine the meaning of codons.
They produced a synthetic RNA by using the enzyme polynucleotide phosphorylase discovered in 1955 by Grunberg. Manago and Ochoa
This enzyme does not use DNA as a template and can incorporate ribonucleotides in a random order.
The first synthetic RNA used for protein in a cell-free system (the in vitro system consists of ribosomes, tRNA’s and other necessary factors for protein synthesis) was poly U i.e., the mRNA consists of only one nucleotide namely U in a series.
This can be called a homopolymer as it is a polymer of only one nucleotide. The poly U mRNA was able to synthesis a homopolymer of a polypeptide chain containing only phenylalanine residues. This proved that the triplet code UUU specifies phenylaline.
Subsequently many such homopolymer species of mRNA were synthesized and were directed to produce polypeptide chains. For example AAA was shown to code for lysine and CCC specified proline.
Nirenberg and Ochoa subsequently used heteropolymeres of mRNA to produce polypeptide chains. A heteropolymer includes a mixture of two, three or all the four nucleotides in mRNA. This yielded further information on the possible codons for certain amino acids.
For example poly CG directs the incorporation of arginine and alanine in addition to the proline coded by poly C.
Some additional information on the assignment of codons to individual amino acids was obtained by varying the ratios of different nucleotides in the synthetic mRNA. In this experiment the code assignment was at best ambiguous since all the four bases were not required for the incorporation of any of the amino acids.
3. Binding of known triplets to aminoacyl tRNA:
A triribonucleotide cannot direct the synthesis of a polypeptide but it leads to binding of specific aminoacyl tRNA to the ribosomes. Nirenberg et al used this property for assigning specific codons of specific amino acids.
Their technique is known Nirenberg – Leder technique. Conventionally a trinucleotide with 5′ terminal phosphate are more active and those lacking this phosphates in inducing the binding of aminoacyl tRNA to ribosome’s.
On the otherhand those with 3′ terminal phosphate are inactive. This shows that the codons must be triplet. In the Nirenberg – Leder technique trinucleotides with 5′ terminal phosphate are added to a cell free system containing a mixture of 20 amino acids bound to their respective tRNA. One of the 20 amino acids had radioactive carbon.
For different experiments different labeled amino acids were used but with the same trinucleotide after the synthesis of protein the cell free mixture containing ribosomes were analysed to detect radioactivity confirming the binding of aminoacyl tRNA to the ribosomes.
These studies showed that UUU, CCC and AAA induced the binding of phenylalnine, proline and lycine respectively to ribosomes. Using this technique Nirenberg et al assigned most of the codons to their respective amino acids.
4. Amino acid sequence generated by mRNA transcribed from synthetic tRNA of know nucleotides sequences:
H.G. Khorana and his associates used the above technique for codon assignment in vitro conditions. Known as Khorana technique in this method long specific DNA sequences are first synthesized.
These are then transcribed to mRNA molecules using the DNA directed RNA polymerase enzyme. These mRNA molecules which have known sequences of nucleotide are directed to synthesize polypeptides.
The polypeptides were then subjected to sequential degradation to know the amino acid sequences. The nucleotides sequence is then compared with the amino acid sequences. The nucleotide sequence is to deduce specific codons for different amino acids.
Using this technique almost all the codons for amino acids were designated. In this technique Khorana and associates also confirmed many other characters of the code such as degenerecy, commaless nature, none overlapping and triplet nature also.