Genetic Code

The sequence of nucleotides in DNA determines the sequence of amino acids found in all proteins. Since there are only four nucleotide "letters" in the DNA alphabet (A, C, G, T, which stand for adenine, cytosine, guanine, and thymine), but there are 20 different amino acids in the protein alphabet, it is clear that more than one nucleotide must be used to specify an amino acid. Even two nucleotides read at a time would not give sufficient combinations (4 × 4 = 16) to encode all 20 amino acids plus start and stop signals. Therefore it would require a minimum of three DNA nucleotides to "spell out" one amino acid, and indeed this is the number that is actually used. RNA also uses a four letter alphabet when it reads and transcribes DNA instructions during protein synthesis, but its set of nucleotides is somewhat different, substituting U (uracil) for T (thymine).

Any single set of three nucleotides is called a codon , and the set of all possible three-nucleotide combinations is called "the genetic code" or "triplet code." There are sixty-four different combinations or codons (4 × 4 × 4 = 64). We now know that three codons (UAA, UAG, and UGA) specify a "stop" signal, indicating the termination of the polypeptide chain being synthesized on the ribosome. Each of the remaining sixty-one codons encodes an amino acid. The "start" signal is the codon AUG, which also encodes the amino acid methionine. The codons are read from the messenger RNA molecule during protein synthesis, and, consequently, they are given in RNA bases rather than in the original DNA sequence. The reading of the codons is shown in Figure 1.

Translation

The gene is represented by the sequences of bases in the DNA molecule, which can, in a sense, be thought of as a "storage molecule" for genetic information. DNA is extremely stable, a property critical to the maintenance of the integrity of the gene. This stability is evidenced by the fact that DNA has been extracted from Egyptian mummies and extinct animals such as the woolly mammoth. It can be extracted from dried blood or from a single hair at a crime scene.

Each cell contains a complete set of genes, but only certain of these genes are active or "expressed" at any one time. When a gene is active, a "disposable" copy is transcribed from the gene into codons contained in a messenger RNA (mRNA) molecule. Unlike the DNA molecule, the mRNA molecule is relatively unstable and short-lived. This is so that when a gene is turned off, the mRNA does not remain in the cell forever, running off more proteins on the ribosomes that are no longer needed by the cell.

Another RNA molecule, called transfer RNA (tRNA), contains a specific region called the anticodon. The tRNA anticodon can base pair with the codon region of the mRNA during protein synthesis, using the base pairing rules of A-U, U-A, C-G, and G-C. Each tRNA carries a specific amino acid. Thus the tRNA carrying methionine has a UAC anticodon that pairs with the AUG codon of the mRNA bound to the ribosome. Similarly the tRNA for proline has a GGA anticodon.

In examining the table of codons (Table 1) you will see that there is more than one codon for each amino acid, except for methionine (AUG) and tryptophan (UGG). Different codons that code for the same amino acid are said to be "synonyms," and the code is said to be "degenerate" in the sense that there is not a single, unique codon for each of the twenty amino acids.

The "Wobble" Hypothesis

Even before the genetic code had been elucidated, Francis Crick postulated that base pairing of the mRNA codons with the tRNA anticodons would require precision in the first two nucleotide positions but not so in the third position (the precise conformation of base pairs , which refers to the hydrogen bonding between A-T (A-U in RNA) and C-G pairs is known as Watson-Crick base pairing). The third position, in general, would need to be only a purine (A or G) or a pyrimidine (C or U). Crick called this phenomenon "wobble."

This less-than-precise base pairing would require fewer tRNA species. For example, tRNA^Glu could pair with either GAA or GAG codons. In looking at the codon table, one can see that, for the most part, the first two letters are important to specify the particular amino acid. The only exceptions are AUG (Met) and UGG (Trp) which, as indicated above, have only one codon each.

The Code Has No Gaps or Overlaps

The 1960s were an exciting time for molecular biologists, for it was then that the genetic code was broken. Two possibilities had to be considered for the genetic code. It was possible that the code had gaps, that is, some sort of punctuation mark or a "spacer" nucleotide or nucleotides between coding groups. Second, the code could be either overlapping or nonoverlapping. These possibilities are illustrated in Figures 2 and 3. An overlapping code would have the advantage that more information could be contained in a smaller space.

However, in overlapping code a mutation that changed one base would lead to the changing of three consecutive amino acids in the protein sequence. Genetic evidence, available even before the code had been deciphered, indicated that a single point mutation, that is, a change in a single nucleotide, affected only one amino acid and thus suggested a nonoverlapping code.

Another possibility was that the code had punctuation marks, that is, a base (indicated by "p" in Figure 3) acting as a comma that would separate each codon. In this situation, if an additional base were inserted into a codon, then only that codon would be affected. In a code without punctuations or gaps, however, insertion of a single nucleotide would result in all codons from that point on being affected. This would in turn change the amino acid sequence in the protein from that point on. Again, genetic evidence ruled out a punctuated code, as base insertions do, in fact, affect the entire protein from the insertion point on, rather than just a single amino acid. This effect is called a frameshift mutation.

In the late 1970s DNA sequencing techniques were developed. A number of proteins had already been sequenced by protein sequencing methods. When the genes for these proteins were cloned and sequenced, the predicted protein sequence could be deduced. Agreement between the DNA and protein sequences confirmed the accuracy of the genetic code.

Exceptions to the Universal Genetic Code

After the original genetic code of E. coli was completed in 1968, the genetic code was subsequently determined for many other organisms ranging from bacteria to mammals, including humans. The codons were found to be the same for all organisms, leading to the idea that the genetic code is "universal." Furthermore, it also suggested that life on Earth had a single evolutionary origin, otherwise there would have been numerous genetic codes. The code was established during evolution, probably by chance, as there are no compelling reasons one codon should prevail over another. After it was established, any subsequent changes in the code would prove to be lethal, for if one codon changed, then all similar codons in the entire organism's genome would have to change simultaneously—a highly unlikely possibility.

Thus, it was surprising to find that there are, in fact, a few rare exceptions to the universal code. These exceptions are listed in Table 2. Most of these exceptions are found in the mitochondrial genome. The mitochondrion is thought to have evolved from an endosymbiotic bacterium at the time when the eukaryotic cell first arose. The mitochondrial genome is small, and most of the genes of the original endosymbiont have migrated to the nucleus.

In examining the exceptions to the universal genetic code in Table 2, you can see that there are only a few changes, most notably the use of a standard "stop" codon to encode an amino acid. For example, UGA normally is a stop codon. But in the mitochondria of the fruit fly Drosophila melanogaster, it encodes the amino acid tryptophan.

A few additional exceptions to the universal genetic code have also been identified. These include the nuclear genome of a few protozoan species and also in the bacterium Mycoplasma capricolum. These exceptions, however, do not imply multiple evolutionary origins of life. What is most striking is that the "exceptional" meanings of most of the codons are identical across all the organisms in which they are found, not different. Had there been multiple origins, we would expect to see drastically different genetic codes in these exceptional organisms.

see also Crick, Francis; Escherichia coli (E. coli bacterium); Nucleotide; Reading Frame; Ribosome; Transcription; Translation.

Ralph R. Meyer

Bibliography

"The Genetic Code." Cold Spring Harbor Symposia on Quantitative Biology, vol. 31. Cold Spring Harbor, NY: Cold Spring Harbor Press, 1966.

Kay, Lily E. Who Wrote the Book of Life? A History of the Genetic Code. Stanford, CA: Stanford University Press, 2000.

Nirenberg, M. W., and J. H. Matthaei. "The Dependence of Cell-Free Protein Synthesis in E. coli upon Naturally Occurring or Synthetic Polyribonucleotides." Proceedings of the National Academy of Sciences 47 (1961): 1588-1602.