Principles of Biochemistry Volume 3 - A. Lehninger 1985

Molecular mechanisms of genetic information transmission
Protein synthesis and its regulation
The genetic code has been deciphered

We will now examine in more detail how the four-letter language of DNA is translated into the twenty-letter language of Proteins. It has long been clear that at least three nucleotide residues of DNA are required to encode each amino acid. Since the four DNA code letters (A, T, G, and C) can form only 16 different pairs (4² = 16), this is not enough to encode 20 Amino Acids. However, combining the four bases in groups of three yields 4³ = 64 different combinations. Early genetic experiments definitively proved not only that the words of METABOLISM/28.html">The Genetic Code for any amino acid consist of nucleotide triplets, but also that there are no punctuation marks between the codons for adjacent amino acids. Nevertheless, a fundamental question remained unanswered: which specific three-letter code words correspond to each of the amino acids? How can this be determined experimentally?

In 1961, Marshall Nirenberg and Heinrich Matthaei reported on an experiment that became a milestone in this field. They incubated synthetic polyribonucleotide polyuridylic acid with an E. coli extract, GTP, and a mixture of twenty amino acids in twenty separate tubes. Only one of the amino acids was radioactively labeled in each tube. Given that polyuridylic acid (designated as polyU) is an artificial mRNA carrying multiple consecutive UUU triplets, it logically followed that it should direct the synthesis of a radioactive polypeptide consisting of a single amino acid—specifically, the one encoded by the UUU triplet. A radioactive polypeptide was synthesized in only one of the twenty tubes: the one containing radioactive phenylalanine. The radioactive polypeptide turned out to be polyphenylalanine, meaning it contained solely phenylalanine residues. From this, Nirenberg and Matthaei concluded that the UUU triplet encodes phenylalanine. Very soon, using a similar approach, it was discovered that the synthetic template polycytidylic acid (polyC) directs the synthesis of a polypeptide consisting entirely of Proline residues (polyproline), while polyadenylic acid (polyA) encodes polylysine. Consequently, the CCC triplet must correspond to proline, and the AAA triplet to Lysine.

The synthetic polynucleotides used in these experiments were produced using polynucleotide phosphorylase (section 28.28), which readily forms RNA-like polymers from ADP, UDP, CDP, and GDP. This enzyme does not require a template; it synthesizes polymers whose nucleotide composition reflects the relative concentrations of the starting nucleoside 5'-diphosphates present in the medium. If the medium contains only uridine diphosphate, polynucleotide phosphorylase synthesizes exclusively polyU. If the initial mixture consists of two parts ADP and one part GDP, a polymer is synthesized in which approximately two-thirds are A residues and one-third are G residues. Such a polymer with a non-specific nucleotide sequence likely contains many AAA triplets, fewer AAG, AGA, and GAA triplets, relatively few AGG, GGA, and GAG triplets, and very few GGG triplets. By employing various synthetic polyribonucleotides generated by polynucleotide phosphorylase from different starting mixtures of ADP, UDP, GDP, and CDP to serve as mRNA, researchers were soon able to determine the triplet composition for all amino acids. However, these experiments did not allow for the identification of The nucleotide sequence within each coding triplet—that is, the exact order of the letters comprising that codon.

In 1964, Nirenberg and Philip Leder made another discovery that led to the resolution of this problem. They found that Ribosomes isolated from E. coli bind specific aminoacyl-tRNA even in the absence of GTP, provided that a corresponding synthetic polynucleotide template is present in the mixture. For example, ribosomes incubated with polyU and phenylalanyl-tRNA bind to both macromolecules; however, if ribosomes are incubated with polyU and any other aminoacyl-tRNA, the latter fails to bind because its anticodon does not recognize the UUU triplet in the polyU template. The shortest polynucleotide capable of ensuring the specific binding of phenylalanyl-tRNA^Phe turned out to be the trinucleotide UUU. Utilizing simple trinucleoids of known Structure, researchers successfully established the nucleotide sequence within the codons that dictate the binding of various aminoacyl-tRNAs. The application of this and other approaches soon made it possible to decipher the nucleotide sequence of all triplets for every amino acid. These code words were verified through numerous independent Methods. The complete amino acid code “dictionary” is presented in Fig. 29-22. The cracking of the genetic code stands as one of the greatest scientific achievements of the 1960s.