The genetic code has been cracked or deciphered by the following kinds of approaches:
A. Theoretical Approach:
George Gamow proposed the diamond code (1954) and the triangle code (1955) and suggested the following properties of the genetic code:
(i) A triplet codon corresponding to one amino acid of the polypeptide chain.
(ii) Direct template translation by codon-amino acid pairing.
(iii) Translation of the code in an overlapping manner.
(iv) Degeneracy of the code, i.e., an amino acid being coded by more than one codon.
(v) Colinearity of nucleic acid and the primary protein synthesized.
(v) Universality of the code, i.e., the code being essentially the same for different organisms.
B. The in vitro codon Assignment:
1. Discovery and use of polynucleotide phosphorylase enzyme:
Manago and Ochoa isolated an enzyme from the bacteria (e. g., Azobacter vinelandii) that catalyzes the breakdown of RNA in bacterial cells. This enzyme is called polynucleotide phosphorylase. They found that outside of the cell (in vitro), with high concentrations of ribonucleotides, the reaction could be driven in reverse and an RNA molecule could be made.
Incorporation of bases into the molecule is random and does not require a DNA template. Thus, in 1955 Manago and Ochoa made possible the artificial synthesis of polynucleotides (=mRNA) containing only a single type of nucleotides (U, A, C, or G respectively) repeated many times.
1. Polyuridylic acid or poly (U)
2. Polyadenylic acid or poly (A)
3. Polycytilic acid or poly (C)
4. Polyguanidylic acid or poly (G)
Thus, the action of polynucleotide phosphorylase can be represented in the following way:
(RNA)n + ribonucleoside diphosphate ß—-àpolynucleotide phosphorylase (RNA)n+1 + Pi
The polynucleotide phosphorylase enzyme differs from RNA polymerase used to transcribe mRNA from DNA polymerase used to transcribe mRNA from DNA in that: (i) it does not require a template or primer; (ii) the activated substrates are ribonucleoside diphosphates (e. g., UDP, ADP, CDP and GDP) and not triphosphates; and (iii) orthophosphate (Pi) is produced instead of pyrophosphates (PPi).
The deciphering of the genetic code was made possible by the use of synthetic (or artificial) polynucleotides and trinucleotides. The different types of techniques used include the use of polymers containing a single type of nucleotide (called homopolymers), the use of mixed polymers (copolymers) containing more than one type of nucleotides (heteropolymers) in random or defined sequences and the use trinucleotides (or “minimessengers”) in ribosome-binding or filter-binding.
2. Codon assignment with unknown sequence:
(i) Codon assignment by homopolymer:
The first clue to codon assignment was provided by Nirenberg and Matthaei (1961) when they used in vitro system for the synthesis of a polypeptide using an artificially synthesized mRNA molecule containing only one type of nucleotide (i.e., homopolymer). Prior to performing the actual experiments, they tested the ability of a cell-free protein synthesizing system to incorporate radioactive amino acids into newly synthesized proteins.
Their cell-free extracts of E. coli contained ribosomes, tRNAs, aminoacyl-tRNA synthetase enzymes, DNA and mRNA. The DNA of this extract was eradicated by the help of deoxyribonuclease enzyme, thus, the template which might synthesize new mRNA was destroyed.
When twenty amino acids were added to this mixture along with ATP, GTP, K+ and MG2+, they were incorporated into proteins. This incorporation continued so long as mRNA was present in such a cell-free suspension. It also continued in the presence of synthetic polynucleotides (mRNAs) which could be made with the help of polynucleotide phosphorylase enzyme.
The first successful use of this technique was made by Nirenberg and Matthaei who synthesized a chain of uracil molecules (poly U) as their synthetic mRNA (homopolymer). Poly (U) seemed a good choice, because there could be no ambiguity in a message consisting of only one base. Poly (U) was good choice for other reasons: it binds well to ribosomes and, as it turned out, the product protein was insoluble and easy to isolate.
When poly (U) was presented as the message to the cell-free system containing all the amino acids, one amino acid was exclusively selected from the mixture for incorporation into the polypeptide, called polyphenylalanine. This amino acid was phenylalanine and it could be concluded that some sequence of UUU coded for phenylalanine. Other homogeneous chains of nucleotides (Poly A, Poly C and Poly G) were inactive for phenylalanine incorporation. The mRNA code word of phenylalanine was, therefore, shown to be UUU. The corresponding DNA code word for phenylalanine can be deduced to be AAA. Thus, the first code word to be deciphered was UUU.
This discovery was extended in the laboratories of Nirenberg and Ochoa. The experiment was repeated using synthetic poly (A) and poly (C) chains, which gave polylysine and polyproline respectively. Thus, AAA was identified as the code for lysine and CCC as code of proline. A poly (G) message was found non-functional in vitro, since it attains secondary structure and, thus, could not attach to the ribosomes. In this way three of 64 codons were easily accounted for.
(ii) Codon assignment by heteropolymers (Copolymers with random sequences):
Further exposition of the genetic code took place by using synthetic messenger RNAs containing two kinds of bases. This technique was used in the laboratories of Ochoa and Nirenberg and led the deduction of the composition of codons for the 20 amino acids. The synthetic messengers contained the bases at random (called random copolymers). For example, in a random copolymer using U and A nucleotides eight triplets are possible, such as UUU, UUA, UAA, UAU, AAA, AAU, AUU and AUA.
Theoretically, eight amino acids could be coded by these eight codons. Actual experiments, however, yielded only six phenylalanine, leucine, tryosine, lysine asparagine and isoleucine. By varying the relative compositions of U and A in the random copolymer and determining the percentage of the different amino acids in the proteins formed, it was possible to deduce the composition of the code for different amino acids.
3. Assignment of codons with known sequences, (i) Use of trinucleotides or minimessengers in filter binding (Ribosome-binding technique):
Ribosome binding technique of Nirenberg and Leder (1964) made use of the finding that aminoacyl-tRNA molecules specifically bind to ribosome-mRNA complex. This binding does not require the presence of a long mRNA molecule; in fact, the association of a trinucleotide or minimessenger with the ribosome is sufficient to cause aminoacyl-tRNA binding.
When a mixture of such small mRNA molecules- ribosomes and amino acid-tRNA complexes are incubated for a short time and then filtered through a nitrocellulose membrane, then the mRNA-ribosome-tRNA-amino acid complex is retained back and rest of the mixture passes through the filter.
By using a series of 20 different amino acid mixtures, each containing one radioactive amino acid at a time, it is possible to find out the amino acid corresponding to each triplet by analysing the radioactivity absorbed by the membrane, e.g., the triplet GCC and GUU retain only alanyl-tRNA and valyl-tRNA respectively. All 64 possible triplets have been synthesized and tested in this way. Forty five of them have given clear-cut results. Later on, with the help of longer synthetic messages it has been possible to decipher 61 out of the possible 64 codons.
C. The in vivo Codon Assignment:
The cell free protein synthetic systems, though have proved of great significance in decipherment of the genetic code, but they could not tell us whether the genetic code so deciphered is used in the living systems of all organisms also. Three kinds of techniques are used by different molecular biologists to determine whether the same code is also used in vivo (a) amino acid replacement studies (e.g., tryptophan synthetase synthesis in E. coli (Yanofsky et al. 1963) and haemoglobin synthesis in man), (b) fraftieshift mutations (e.g., investigations of Terzaghi et. al. 1966, on lysozyme enzyme of T4 bacteriophages, and (c) comparison of a DNA or mRNA polynucleotide cryptogram with its corresponding polypeptide clear text (e.g., comparison of amino acid sequence of the R17 bacteriophage coat protein with the nucleotide sequence of the R17 mRNA in the region of the molecule that dictates coat-protein synthesis by S. Cory et. al., 1970).
Thus, in vitro and in vivo studies, so far described, gave the way to formulate a code table for twenty amino acids.