The triplet code experiment

Specific nucleotide sequences make up genes. Therefore genes make up codes. This concept can be made simpler with the help of languages. There are different languages spoken by humans. However, all the humans cannot understand all the languages. The words used in a language are like codes. Only the person who knows to speak that language understands those words. Just as words define a language, the genes use codes for communication and sending signals. Hence a genetic code is a linear sequence of nucleotides that specify amino acids. The genetic code is a triplet code. Two popular experiments explaining the theory of triplet code are Francis Brenner experiment and Nirenberg Khorana experiment respectively. Any changes in the sequence of nucleotides lead to different types of mutations, thereby affecting the structure and synthesis of the proteins. There are specific codons for the initiation of protein synthesis, and many such functions. Also, there are two schools of thoughts regarding the universality of the genetic code. Few references suggest the universal nature of the genetic code whereas other concepts contradict this concept.

Insertion or deletion of a letter changes the meaning of the word. Fear becomes far due to deletion of a letter. Changes in a word could change the meaning of the sentence. Consider the letters as nucleotides, the words as amino acids and the sentence as the polypeptide. Following experiments describe the triplet code.

Image: The triplet code experiment

1.   Crick-Brenner Experiment

The evidence of the triplet code came from the experiments on T₄ phage carried out by Francis Crick and his colleagues. The T₄ bacteriophage is a virulent phage that undergoes lytic cycle. The phage infects the E. coli cells, breakdowns the bacterial chromosomes, replicates itself, produces its progeny and finally lyses the cells. Francis Crick decided to use the mutants and wild-type phages. The mutants and wild-type strains are known as rII and r+ strains respectively. The rII mutants are known to produce clear plaques whereas the wild-type phages produce turbid plaques. The first step of the experiment involved the creation of mutations in the wild-type strains. A mutagen known as proflavin was used to induce mutations. A series of addition-deletion steps were carried out. Addition or deletion of base pairs led to frameshift mutations. The mutagen was capable of reverting a mutant strain to wild-type. The process of reverting a mutant to wild-type strain is known as reversion. Reversion of addition mutations was possible with a deletion of a base pair. Addition of a base pair resulted into reversion of a deletion mutation. Different rII mutations were combined to check the reversions. Three nearby mutations gave rise to the revertants. No other combinations worked. Therefore they concluded that the genetic code is a triplet code.

2.   Nirenberg- Khorana experiment of deciphering the genetic code:

Marshall Nirenberg and Gobind Khorana with Robert Holley shared a Nobel prize in physiology and medicine for deciphering the genetic code. They established an exact relationship between the 64 codons and 20 different amino acids through an extensive project. The basis of the experimentation was a cell-free protein synthesizing system. This system consisted of components isolated and purified from E. coli bacteria. The components in this system were ribosomes, tRNA with attached amino acids, and protein factors. In addition to these molecules, radioactively labeled amino acids were incorporated. It was essential to determine the nature of the genetic code, the codons, and their specificity to express the amino acid. Preparation and addition of synthetic mRNAs to the system helped to analyze the polypeptides. The following table describes the results.

Type of synthetic mRNA	Polypeptide chain with an amino acid
Poly (U) mRNA	Phenylalanine
Poly (A) mRNA	Lysine
Poly (C) mRNA	Proline
Poly (G) mRNA	Inconclusive result (Poly (G) folds up)

Table: Different types of synthetic mRNAs with their corresponding polypeptide chains having specific amino acids.

Next step was to analyze synthetic mRNA with two incorporated bases. Such molecules are known as random copolymers. The poly (AC) molecules revealed eight different codons such as CCC, CCA, CAC, ACC, CAA, ACA, AAC, and AAA. So the poly (AC) mRNA resulted into polypeptide chain with asparagine, glutamine, histidine, and threonine in addition to lysine and proline. Amino acid incorporation is based on the ratio of both the bases. Arginine was added to the polypeptide on an increased number of adenines in comparison with the cytosines. A histidine was incorporated into the polypeptide if cytosines exceeded the adenines.

The third experimental approach involved synthesized copolymers instead of random copolymers.

The advantage of a synthesized copolymer was that its sequence was known. It was pre-tested in a cell-free protein synthesizing system. The polypeptide consisted of repeating amino acid patterns.

The fourth experimental design utilized a ribosome binding assay. The ribosome forms complex with specific RNA molecules. Thus it helped to conclude the specific relationships between many codons and amino acids for which they code.

Characteristics of the genetic code

1.     Since there are three nucleotides in a codon, the genetic code is a triplet code. The phage rII mutants reverted to wild-type after adding or deleting three nucleotide base pairs.

2.     There is a continuous reading of three nucleotide base pairs indicating the continuous nature of the genetic code.

3.     There is no overlapping of the triplet code.

4.     The code is almost universal except mitochondrial genomes in few species.

5.     The code is degenerate since one code can generate more than a single amino acid.

6.     Start codons initiate the process of translation and stop codons terminate the process.

7.     There is a wobble in the genetic code. The tRNA may follow wobble pairing with the 3’ end base of certain codons.

   References:
[1] Genomes, T.A. Brown, third edition
[2] Crick, Brenner et al. experiment - Wikipedia
[3] Nirenberg and Leder experiment - Wikipedia

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