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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64 Codons

DNA, the genetic material, replicates and undergoes a process known as transcription to synthesize RNA molecules. The genes or small regions in the DNA code for RNA. Four nucleotides constitute the double helix structure. Adenine, guanine, thymine, and cytosine are nucleotide bases present in the DNA structure. An RNA molecule consists of uracil instead of thymine. RNA is mainly involved in synthesizing proteins. Primary to quaternary structures of proteins are amino acid-dependent. Therefore, proteins also have sequences like DNA and RNA. Nucleic acids have gene sequences while proteins have amino acid sequences. Most of the proteins are not self-synthesizing because they are RNA dependent. A, C, G and U in an RNA molecule are not just letters but are capable of creating huge sentences. Meaning, A, C, G, and U nucleotides together form three-letter codes to generate codons. Thus an amino acid sequence is specified by these four nucleotides.

The concept of the codon is very simple to understand. Imagine any three of the above nucleotides together, say AUG. Thus, AUG becomes a codon. It can code for an amino acid or a signal. AUG is known as an initiation codon. It is involved in the initiation of translation (protein synthesis). Thus three nucleotide bases come together in triplets and generate total 64 codons. Why only three letter code? The logic behind cells utilizing three letter code instead of one or two letter code is to create 20 different amino acids. Just one or two letter codes won’t be sufficient. A code with more than three bases would possibly synthesize undesirable products. Thus a three letter code fits perfect, in the picture.

Image 1: Codon dictionary

The discovery of a triplet code:

Francis Crick and his colleagues worked on T₄ bacteriophage and discovered the triplet code of genes. Note that T₄ is a virulent phage. This phage produces its progeny in the E. coli cells and releases them through cell lysis. The Crick Brenner experiment demonstrated the triplet nature of the genetic code. They used a mutagen known as proflavin which was capable of inserting or deleting a base pair causing frameshift mutations. The experimenters tried adding and deleting the base pairs. After many such attempts, they realized the gene function to be dependent on three base pairs. Thus, they concluded that the genetic code uses a codon of three nucleotide bases. They used mutant and wild-type strains of phage. The rII phages exhibit mutant phenotypes and r+ phages exhibit wild-type phenotype. The reversion of mutant to wild-type is possible with proflavin. A base pair was added to reverse the deletion mutation. Later on, the experiments were carried out by Nirenberg and Gobind Khorana. They established an exact relationship between 64 codons and 20 amino acids. They used cell-free, protein synthesizing system with purified components isolated from E. coli. They consisted of ribosomes, tRNAs and protein factors. The study was highly extensive since the aim was to find out which codon specified for which amino acid. So they prepared synthetic mRNAs with different types of bases and were added to the system. Hence they utilized different copolymers to decipher the genetic code.

Characteristics of the genetic code:

1.     The genetic code is a triplet code. It means there are three nucleotides in a codon.

2.     The code is continuous because the mRNA is continuously read. Three nucleotides are read at a time without an erroneous skip.

3.     The triplet code does not overlap. The code reads the mRNA in successive groups of three nucleotides.

4.     The code is almost universal. The genetic language shared by all the organisms is almost the same.

5.     The code exhibits degeneracy. It means that one code can generate more than one amino acid.

6.     The code has start and stop signals.

7.     There is a wobble in the genetic code. 

INFO-BOX: Terminologies

Code: It is a sequence of nucleotides. Codon: It is a section of DNA consisting of three nucleotide pairs or a section of RNA consisting of a code for a single amino acid. Code dictionary: It is a listing of 64 possible codons and their translational meanings. Coding region: It is an open reading frame. A coding region is also known as Exon that encodes for a protein. Coding strand: It is a DNA strand consisting of the same sequence as a transcribed mRNA with a linear array of codons. These codons interact with the anti-codons so that they can give a primary sequence of a protein. Codon preference: This concept deals with a disproportionate usage of codons. They would correspond with an abundance of tRNAs.

Wobble Hypothesis:

As discussed earlier, there are 64 codons. 61 out of 64 codons are sense codons. The remaining three codons are known as non-sense codons or stop codons. Francis Crick proposed the wobble hypothesis. The hypothesis states that the tRNAs are capable of reading the codons. The 5’ end base of the anticodon is not as constrained as the other two bases. For example, consider two leucine codons such as CUC and CUU. Leucine tRNA can read these two codons. Leucine tRNA normally pairs with CUC since it has a GAG sequence. However while pairing with CUU, the leucine tRNA follows Wobble pairing. The presence of modified purine inosine at its 5’ end of the anticodon enables it to recognize three different codons.

Image 2: Wobble pairing

The genetic code may not necessarily be universal:

Originally it was considered that the genetic code is universal. It won't be possible to change an established code. However, the genetic code may not be universal. There are deviations in some organisms. The mitochondrial genomes in some organisms use non-standard codes. For example, the mitochondrial genome of mammals exhibits UGA codon which codes for tryptophan instead of the stop codon. The same UGA codon codes for cysteine in Euplotes species. Various context-dependent codon reassignments are present in archaea. For example archeal UGA codon codes for selenocysteine and UAG codes for pyrrolysine. Non-standard codes are also known for nuclear genome of lower eukaryotes and may involve reassignment of termination codons.

Codon usage bias:

A synonymous codon frequency in a coding DNA is known as codon usage bias. A synonymous substitution is evolutionary and substitutes one base for another in a coding region. There could be a greater frequency of coding the same amino acid as if biased. Codon biases try to create a balance between mutational biases and natural selection for translation optimization. The gene expression levels, the G-C compositions, strand-specific mutational bias, GC skew, and many other factors contribute towards codon usage bias. 

References:

[1] Genomes, T.A. Brown, third edition

[2] Human molecular genetics 3, T. Strachan, Andrew P. Read, Volume 3
[3] A textbook of biotechnology, R.C. Dubey

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