In this article we will discuss about the approaches made for codon assignments.
Contents
1. Codon Assignment with Unknown Sequences:
Theoretically it was considered that genetic code should be triplet so that codons must be assigned for 20 amino acids. But it was not known which codon synthesizes which amino acid. Therefore, the first approach was made to find out the codons for amino acids by using sequences of mRNA in vitro, and secondly to prove the same in vivo.
(i) Use of Homopolymers:
In the first approach, Nirenberg and Matthaei (1961) synthesized the RNA by using polyuridylic acid (poly-U). This resulted in poly-U along the length of mRNA with possible triple UUU codon. When poly-U (RNA) was used for cell free synthesis of polypeptide by using cell free extract of E. coli that supplied all the components of protein synthesizing machinery, polyphenylalanine was synthesised.
It means UUU triplet coded for amino acid phenylalanine. This news about cracking the genetic code was brought out in news papers throughout the world in 1961. Later on they discovered that poly-C induced the synthesis of proline, therefore, CCC triplet was assigned to proline.
Poly-A stimulated polylysine synthesis. Hence, the sequence of AAA designates lysine. The experiments with poly-G were unsuccessful because it did not attach to ribosome due to attaining the secondary structures.
(ii) Use of Copolymers:
In the next experiment, Nirenberg and co-workers used artificially synthesised random ribopolynucleotide containing two or three different nucleotides. For example, if we use A and C we will get poly AC. One can calculate the possible combinations in mixed copolymers. From a mixture of A and C, eight possible triple codons (e.g. AAA, AAC, ACA, ACC, CAC, CCC, CCA and CAA) with equal frequency are formed.
However, when known amount of A and C is used in synthesis, the proportion of eight codons can be calculated. For example, if A: C = 5:1 (i.e. 5/6 is A and 1/6 is C), the eight possible combinations would be as shown in Table 7.2.
The proportion of amino acids coming out of calculated relative proportion of codons synthesised by poly AC were calculated (Table 7.2). Initially the codons were assigned on the basis of base composition, but not sequences of bases in codons.
Hence, by using poly AC six amino acids viz, lysine, asparagine, threonine, glutamine, proline and histidine were found in polypeptide of AC (Table 7.3). However, if the ratio of poly A was more than poly C, the ratio of asparagine to histidine increased accordingly.
2. Codon Assignment with Known Sequences:
The codons assigned to amino acids were found out and a dictionary of codon composition was prepared by using a large number of copolymers of unknown sequences. The next approach was to search out the sequence of codons of known composition.
(i) Codon Assignment through Fitter Binding Technique:
In 1964, Leder and Nirenberg (1964) found when simple trinucleotides of known sequence (i.e. with 5′ and 3′ ends’) are added to ribosomes, it causes the ribosome to attach it to only aminoacyl- tRNA that contains anticodon complementary to trinucleotide (mixed with reaction mixture).
Codon + ribosome + AA- tRNA → Ribosome – codon – AA- tRNA
For example, the trinucleotide GCC is activated to bind alanyl- tRNA but not aminoacyl-tRNA. This shows that GCC is a codon for alanine but not for others.
In another approach it was also found out that ribosome-codon-AA-tRNA complex absorbed on nitrocellulose membrane when passed through it. Nirenberg and coworkers thought when only one amino acid in alternate manner is made radioactive, mixed with rest of 19 amino acids, codons of known sequences, ribosomes and tRNA, and passed through nitrocellulose membrane, then the presence or absence of radioactivity is detected on the basis of adsorption of complex on membrane.
The same codon is used at all the time. Radioactivity will be observed on the membrane only when the radioactive amino acid takes part in complex formation. Otherwise there would be no radioactivity. Each amino acid is radiolabelled in each sample with 19 non-radioactive amino acids.
Nirenberg and coworkers incorporated the radioactive amino acids in cell free system for protein synthesis containing known sequence of codon (mRNA). The cell free system for protein synthesis contained ribosomes, enzymes, tRNA, etc. which was separated from the remainder of broken E. coli cells by gentle centrifugation. During this process mRNAs are broken, therefore, artificial mRNA of known sequence was incorporated.
The artificial mRNA was prepared by using each nitrogen base and an enzyme polynucleotide phosphorylase obtained from Azotobacter vinelandii or Micrococcus lysodeikticus. Unlike RNA polymerase, the poly nucleotide phosphorylase does not require DNA as template.
In this way Nirenberg constructed polyuridylic acid (UUU…), polycytidylic acid (CCC…), polyadenylic acid (AAA…) and polyguanyl acid (GGG…). The experiment with poly-G was unsuccessful because it did not attach to ribosome. By this technique the cracked 45 codons for amino acids are arginine, alanine, cysteine glutamine, glycine, isoleucine, leucine, methionine, proline, tryptophane, tyrosine, serine and valine.
(ii) Use of Copolymers of Repetitive Sequences:
Almost at the same time Har Govind Khorana and coworkers succeeded in determining the exact sequence of nucleotides in many codons discovered previously by Nirenberg or himself. Khorana prepared artificial mRNA with known sequences and used to ascertain the structural isomer of a codon which specified an amino acid.
For example, if CU (two bases) is repeatedly present throughout the length, it will give the sequence CUCUCUCU In the same way the three bases (ACU) will form the repeatitive sequence ACU ACU ACU ACU Thus, an alternating copolymer e.g. UGU-GUG-UGU-GUG…. was synthesised and used as mRNA in vitro in protein synthesizing system.
Consequently the alternate codons, UGU-GUG-UGU…, synthesised the alternating polypeptide, cysteine-valine-cysteine…. This confirms that both UGU (cysteine) and UUG (leucine) are different codons though base composition is 2U1G.
Similarly, GUG (valine), UGG (tryptophan) and GGU (glycine) are the different codons though all the three have base composition 2G1U. In this way a dictionary of complete genetic code could be prepared (Table 7.4).
3. In Vivo Studies for Codon Assignment:
After carrying out experimentation in vitro on cracking of genetic code it could not provide evidence, whether the genetic code is for all organisms. Therefore, different molecular biologists by using different techniques proved that the same code is also used in vivo as well.
It could be possible through:
(i) Amino acid replacement studies (tryptophan synthetase synthesis in E. coli),
(ii) Frameshift mutations on lysozyme of T4 bacteriophage, and
(iii) Comparison of a DNA or mRNA polynucleotide cryptogram with its corresponding polypeptide.
On the basis of in vitro and in vivo studies, the genetic code could be formulated for all essential amino acids. Each codon is written in genetic dictionary in such a way that appears in mRNA sequence in 5’→ 3′ direction (Table 7.4). In DNA each codon would be complementary and in the reverse order to mRNA on a 5′ → 3′ strand.