CRACKING THE GENETIC CODE
y the early 1960s molecular biologists had adopted the so-called “central dogma,” which states that DNA directs synthesis of RNA (transcription),
which then directs assembly of proteins (translation). However, researchers still did not completely understand how the “code” embodied in DNA and subsequently in RNA directs proteinsynthesis. To elucidate this process, Marshall Nirenberg embarked upon a series of studies that would lead to solution of the genetic code.
Proteins are made from combinations of 20 different amino acids. The genes that encode proteins—that is, specify the type and linear order of their component amino acids—are located in DNA, a polymer made up of only four different nucleotides. TheDNA code is transcribed into RNA, which is also composed of four nucleotides. Nirenberg’s studies were premised on the hypothesis that the nucleotides in RNA form “codewords,” each of which corresponds to one of the amino acids found in protein. During protein synthesis, these codewords are translated into a functional protein. Thus, to understand how DNA directs protein synthesis, Nirenberg setout to understand the relationship between RNA codewords and protein synthesis. At the outset of his studies, much was already known about the process of protein synthesis, which occurs on ribosomes. These large ribonuleoprotein complexes can bind two different types of RNA: messenger RNA (mRNA), which carries the exact protein-specifying code from DNA to ribosomes, and smaller RNA molecules nowknown as transfer RNA (tRNA), which deliver amino acids to ribosomes. tRNAs exist in two forms: those that are covalently attached to a single amino acid, known as amino-acylated or “charged” tRNAs, and those that have no amino acid attached called “uncharged” tRNAs. After binding of the mRNA and the amino-acylated tRNA to
the ribosome, a peptide bond forms between the amino acids, beginningprotein synthesis. The nascent protein chain is elongated by the subsequent binding of additional tRNAs and formation of a peptide bond between the incoming amino acid and the end of the growing chain. Although this general process was understood, the question remained: How does the mRNA direct protein synthesis? When attempting to address complex processes such as protein synthesis, scientists dividelarge questions into a series of smaller, more easily addressed questions. Prior to Nirenberg’s study, it had been shown that when phenylalanie charged tRNA was incubated with ribosomes and polyuridylic acid (polyU), peptides consisting of only phenylalanine were produced. This finding suggested that the mRNA codeword, or codon, for phenylalanine is made up of the nucleosides containing the baseuracil. Similar studies with polycytadylic acid (polyrC) and polyadenylic acid (polyrA) showed these nucleosides containing the bases cytadine and adenine made up the codons for proline and lysine, respectively. With this knowledge in hand, Nirenberg asked the question: What is the minimum chain length required for tRNA binding to ribosomes? The system he developed to answer this question wouldgive him the means to determine which aminoacylated tRNA would bind which m-RNA codon, effectively cracking the genetic code.
The first step in determining the minimum length of mRNA required for tRNA recognition was to develop an assay that would detect this interaction. Since previous studies had shown that ribosomes bind mRNA and tRNA simultaneously, Nirenberg reasoned thatribosomes could be used as a bridge between a known mRNA codon and a known tRNA. When the three components of protein synthesis are incubated together in vitro, they should form a complex. After devising a method to detect this complex, Nirenberg could then alter the size of the mRNA to determine the minimum chain length required for tRNA recognition. Before he could begin his experiment, Nirenberg...