The invention: A method for synthesizing the biological molecule RNA established that this process can occur outside the living cell.
The people behind the invention:
Severo Ochoa (1905-1993), a Spanish biochemist who shared
the 1959 Nobel Prize in Physiology or Medicine Marianne Grunberg-Manago (1921- ), a French biochemist Marshall W. Nirenberg (1927- ), an American biochemist who won the 1968 Nobel Prize in Physiology or Medicine Peter Lengyel (1929- ), a Hungarian American biochemist
RNA Outside the Cells
In the early decades of the twentieth century, genetics had not been experimentally united with biochemistry. This merging soon occurred, however, with work involving the mold Neurospora crassa. This Nobel award-winning work by biochemist Edward Lawrie Tatum and geneticist George Wells Beadle showed that genes control production of proteins, which are major functional molecules in cells. Yet no one knew the chemical composition of genes and chromosomes, or, rather, the molecules of heredity.
The American bacteriologist Oswald T. Avery and his colleagues at New York’s Rockefeller Institute determined experimentally that the molecular basis of heredity was a large polymer known as de-oxyribonucleic acid (DNA). Avery’s discovery triggered a furious worldwide search for the particular structural characteristics of DNA, which allow for the known biological characteristics of genes.
One of the most famous studies in the history of science solved this problem in 1953. Scientists James D. Watson, Francis Crick, and Maurice H. F. Wilkins postulated that DNA exists as a double helix. That is, two long strands twist about each other in a predictable pattern, with each single strand held to the other by weak, reversible linkages known as “hydrogen bonds.” About this time, researchers recognized also that a molecule closely related to DNA, ribonucleic acid (RNA), plays an important role in transcribing the genetic information as well as in other biological functions.
Severo Ochoa was born in Spain as the science of genetics was developing. In 1942, he moved to New York University, where he studied the bacterium Azobacter vinelandii. Specifically, Ochoa was focusing on the question of how cells process energy in the form of organic molecules such as the sugar glucose to provide usable biological energy in the form of adenosine triphosphate (ATP). With postdoctoral fellow Marianne Grunberg-Manago, he studied enzymatic reactions capable of incorporating inorganic phosphate (a compound consisting of one atom of phosphorus and four atoms of oxygen) into adenosine diphosphate (ADP) to form ATP.
One particularly interesting reaction was followed by monitoring the amount of radioactive phosphate reacting with ADP. Following separation of the reaction products, it was discovered that the main product was not ATP, but a much larger molecule. Chemical characterization demonstrated that this product was a polymer of adenosine monophosphate. When other nucleocide diphos-phates, such as inosine diphosphate, were used in the reaction, the corresponding polymer of inosine monophosphate was formed. Thus, in each case, a polymer (a long string of building-block units) was formed. The polymers formed were synthetic RNAs, and the enzyme responsible for the conversion became known as “polynucleotide phosphorylase.” This finding, once the early skepticism was resolved, was received by biochemists with great enthusiasm because no technique outside the cell had ever been discovered previously in which a nucleic acid similar to RNA could be synthesized.
Learning the Language
Ochoa, Peter Lengyel, and Marshall W. Nirenberg at the National Institute of Health took advantage of this breakthrough to synthesize different RNAs useful in cracking the genetic code. Crick had postulated that the flow of information in biological systems is from DNA to RNA to protein. In other words, genetic information contained in the DNA structure is transcribed into complementary RNA structures, which, in turn, are translated into the protein. Protein synthesis, an extremely complex process, involves bringing a type of RNA, known as messenger RNA, together with amino acids and huge cellular organelles known as ribosomes.
Yet investigators did not know the nature of the nucleic acid alphabet—for example, how many single units of the RNA polymer code were needed for each amino acid, and the order that the units must be in to stand for a “word” in the nucleic acid language. In 1961, Nirenberg demonstrated that the polymer of synthetic RNA with multiple units of uracil (poly U) would “code” only for a protein containing the amino acid phenylalanine. Each three units (U’s) gave one phenylalanine. Therefore, genetic words each contain three letters. UUU translates into phenylalanine. Poly A, the first polymer discovered with polynucleotide phosphorylase, was coded for a protein containing multiple lysines. That is, AAA translates into the amino acid lysine.
The words, containing combinations of letters, such as AUG, were not as easily studied, but Nirenberg, Ochoa, and Gobind Khorana of the University of Wisconsin eventually uncovered the exact translation for each amino acid. In RNA, there are four possible letters (A, U, G, and C) and three letters in each word. Accordingly, there are sixty-four possible words. With only twenty amino acids, it became clear that more than one RNA word can translate into a given amino acid. Yet, no given word stands for any more than one amino acid. A few words do not translate into any amino acid; they are stop signals, telling the ribosome to cease translating RNA.
The question of which direction an RNA is translated is critical. For example, CAA codes for the amino acid glutamine, but the reverse, AAC, translates to the amino acid asparagine. Such a difference is critical because the exact sequence of a protein determines its activity—that is, what it will do in the body and therefore what genetic trait it will express.
Consequences
Synthetic RNAs provided the key to understanding the genetic code. The genetic code is universal; it operates in all organisms, simple or complex. It is used by viruses, which are nearly life but are not alive. Spelling out the genetic code was one of the top discoveries of the twentieth century. Nearly all work in molecular biology depends on this knowledge.
The availability of synthetic RNAs has provided hybridization tools for molecular geneticists. Hybridization is a technique in which an RNA is allowed to bind in a complementary fashion to DNA under investigation. The greater the similarity between RNA and DNA, the greater the amount of binding. The differential binding allows for seeking, finding, and ultimately isolating a target DNA from a large, diverse pool of DNA—in short, finding a needle in a haystack. Hybridization has become an indispensable aid in experimental molecular genetics as well as in applied sciences, such as forensics.
See also Artificial chromosome; Artificial hormone; Cloning; Genetic “fingerprinting”; Genetically engineered insulin; In vitro plant culture; Synthetic amino acid; Synthetic DNA.
