The biochemical nuts and bolts of DNA replication were being analyzed at around the same time in Arthur Romberg's laboratory at Washington University in St. Louis. By developing a new, "cell-free" system for DNA synthesis, Kornberg discovered an enzyme (DNA polymerase) that links the DNA components and makes the chemical bonds of the DNA backbone. Romberg's enzymatic synthesis of DNA was such an unanticipated and important event that he was awarded the 1959 Nobel Prize in Physiology or Medicine, less than two years after the key experiments (see Plate 20). After his prize was announced, Romberg was photographed holding a copy of the double helix model I had taken to Cold Spring Harbor in 1953.
It was not until 1962 that Francis Crick, Maurice Wilkins, and I were to receive our own Nobel Prize in Physiology or Medicine. Four years earlier, Rosalind Franklin had died of ovarian cancer at the tragically young age of thirty-seven. Before then Crick had become a close colleague and a real friend of Franklin's. Following the two operations that would fail to stem the advance of her cancer, Franklin convalesced with Crick and his wife, Odile, in Cambridge.
It was and remains a long-standing rule of the Nobel Committee never to split a single prize more than three ways. Had Franklin lived, the problem would have arisen whether to bestow the award upon her or Maurice Wilkins. The Swedes might have resolved the dilemma by awarding them both the Nobel Prize in Chemistry that year. Instead, it went to Max Perutz and John Kendrew, who had elucidated the three-dimensional structures of hemoglobin and myoglobin respectively.
The discovery of the double helix sounded the death knell for vitalism. Serious scientists, even those religiously inclined, realized that a complete understanding of life would not require the revelation of new laws of nature. Life was just a matter of physics and chemistry, albeit exquisitely organized physics and chemistry. The immediate task ahead would be to figure out how the DNA-encoded script of life went about its work. How does the molecular machinery of cells read the messages of DNA molecules? As the next chapter will reveal, the unexpected complexity of the reading mechanism led to profound insights into how life first came about.
CHAPTER THREE
READING THE CODE:
BRINGING DNA TO LIFE
Long before Oswald Avery's experiments put DNA in the spotlight as the "transforming principle," geneticists were trying to understand just how the hereditary material – whatever it might be – was able to influence the characteristics of a particular organism. How did Mendel's "factors" affect the form of peas, making them either wrinkled or round?
The first clue came around the turn of the century, just after the rediscovery of Mendel's work. Archibald Garrod, an English physician whose slow progress through medical school and singular lack of a bedside manner had ensured him a career in research rather than patient care at St. Bartholomew's Hospital in London, was interested in a group of rare diseases of which a common marked symptom was strangely colored urine. One of these diseases, alkaptonuria, has been dubbed "black diaper syndrome" because those afflicted with it pass urine that turns black on exposure to air. Despite this alarming symptom, the disease is usually not lethal, though it can lead in later life to an arthritis-like condition as the black-urine pigments accumulate in the joints and spine. Contemporary science attributed the blackening to a substance produced by bacteria living in the gut, but Garrod argued that the appearance of black urine in newborns, whose guts lack bacterial colonies, implied that the substance was produced by the body itself. He inferred that it was the product of a flaw in the body's chemical machinery, an "error in metabolism" in his words, suggesting there might be a critical glitch in some biochemical pathway.
Garrod further observed that alkaptonuria, though very rare in the population as a whole, occurred more frequently among children of marriages between blood relatives. In 1902, he was able to explain the phenomenon in terms of Mendel's newly rediscovered laws. Here was the pattern of inheritance to be expected of a rare recessive gene: two first cousins, say, have both received a copy of the "alkaptonuria" gene from the same grandparent, creating a one-in-four chance that their union will produce a child homozygous for the gene (i.e., a child with two copies of the recessive gene) who will therefore develop alkaptonuria. Combining his biochemical and genetic analyses, Garrod concluded that alkaptonuria is an "inborn error in metabolism." Though nobody really appreciated it at the time, Garrod was thus the first to make the causal connection between genes and their physiological effect. Genes in some way governed metabolic processes, and an error in a gene – a mutation – could result in a defective metabolic pathway.
The next significant step would not occur until 1941, when George Beadle and Ed Tatum published their study of induced mutations in a tropical bread mold. Beadle had grown up outside Wahoo, Nebraska, and would have taken over the family farm had a high-school science teacher not encouraged him to consider an alternative career. Through the thirties, first at Caltech in association with T. H. Morgan of fruit fly fame and then at the Institut de Biologie Physico-Chimique in Paris, Beadle had applied himself to discovering how genes work their magic in affecting, for example, eye color in fruit flies. Upon his arrival at Stanford University in 1937, he recruited Tatum, who joined the effort against the advice of his academic advisers. Ed Tatum had been both an undergraduate and graduate student at the University of Wisconsin, doing studies of bacteria that lived in milk (of which there was no shortage in the Cheese State). Though the job with Beadle might be intellectually challenging, Tatum's Wisconsin professors counseled in favor of the financial security to be found in a career with the dairy industry. Fortunately for science, Tatum chose Beadle over butter.
Beadle and Tatum came to realize that fruit flies were too complex for the kind of research at hand: finding the effect of a single mutation in an animal as complicated as Drosophila would be like looking for a needle in a haystack. They chose instead to work with an altogether simpler species, Neurospora crassa, the orange-red mold that grows on bread in tropical countries. The plan was simple: subject the mold to X rays to cause mutations – just as Muller had done with fruit flies – and then try to determine the impact of the resulting mutations on the fungi. They would track the effects of the mutations in this way: Normal (i.e., unmutated) Neurospora, it was known, could survive on a so-called minimal culture medium; on this basic "diet" they could evidently synthesize biochemically all the larger molecules they required to live, constructing them from the simpler ones in the nutrient medium. Beadle and Tatum theorized that a mutation that knocked out any of those synthetic pathways would result in the irradiated mold strain being unable to grow on minimal medium; that same strain should, however, still manage to thrive on a "complete" medium, one containing all the molecules necessary for life, like amino acids and vitamins. In other words, the mutation preventing the synthesis of a key nutrient would be rendered harmless if the nutrient were available directly from the culture medium.
Beadle and Tatum irradiated some five thousand specimens, then set about testing each one to see whether it could survive on minimal medium. The first survived fine; so did the second, and the third . . . It was not until they tested strain number 299 that they found one that could no longer exist on minimal medium, though as predicted it could survive on the complete version. Number 299 would be but the first of many mutant strains that they would analyze. The next step was to see what exact capacity the mutants had lost. Maybe 299 could not synthesize essential amino acids. Beadle and Tatum tried adding amino acids to the minimal medium, but still 299 failed to grow. What about vitamins? They added a slew of them to the minimal medium, and this time 299 thrived. Now it was time to narrow the field, adding each vitamin individually and then gauging the growth response of 299. Niacin didn't work, nor riboflavin, but when they added vitamin B6, 299 was able to survive on minimal medium. 299's X-ray-induced mutation had somehow disrupted the synthetic pathway involved in the production of B6. But how? Knowing that biochemical syntheses of this kind are governed by protein e
nzymes that promote the individual incremental chemical reactions along the pathway, Beadle and Tatum suggested that each mutation they discovered had knocked out a particular enzyme. And since mutations occur in genes, genes must produce enzymes. When it appeared in 1941, their study inspired a slogan that summarized what had become the understanding of how genes work: "One gene, one enzyme."
But since all enzymes were then thought to be proteins, the question soon arose whether genes also encoded the many cellular proteins that were not enzymes. The first suggestion that genes might provide the information for all proteins came from Linus Pauling's lab at Caltech. He and his student Harvey Itano studied hemoglobin, the protein in red blood cells that transports oxygen from the lung to metabolically active tissues, like muscle, where it is needed. In particular, they focused on the hemoglobin of people with sickle-cell disease, also known as sickle-cell anemia, a genetic disorder common in Africans, and therefore among African Americans as well. The red blood cells of sickle-cell victims tend to become deformed, assuming a distinctive "sickle" shape under the microscope, and the resulting blockages in capillaries can be horribly painful, even lethal. Later research would uncover an evolutionary rationale for the disease's prevalence among Africans: because part of the malaria parasite's life cycle is spent in red blood cells, people with sickle-cell hemoglobin suffer less severely from malaria. Human evolution seems to have struck a Faustian bargain on behalf of some inhabitants of tropical regions: the sickle-cell affliction confers some protection against the ravages of malaria.
Itano and Pauling compared the hemoglobin proteins of sickle-cell patients with those of non-sickle-cell individuals and found that the two molecules differed in their electrical charge. Around that time, the late forties, geneticists determined that sickle-cell disease is transmitted as a classical Mendelian recessive character. Sickle-cell disease, they therefore inferred, must be caused by a mutation in the hemoglobin gene, a mutation that affects the chemical composition of the resultant hemoglobin protein. And so it was that Pauling was able to refine Garrod's notion of "inborn errors of metabolism" by recognizing some to be what he called "molecular diseases." Sickle-cell was just that, a molecular disease.
In 1956, the sickle-cell hemoglobin story was taken a step further by Vernon Ingram, working in the Cavendish Laboratory where Francis Crick and I had found the double helix. Using recently developed methods of identifying the specific amino acids in the chain that makes up a protein, Ingram was able to specify precisely the molecular difference that Itano and Pauling had noted as affecting the overall charge of the molecule. It amounted to a single amino acid: Ingram determined that glutamic acid, found at position 6 in the normal protein chain, is replaced, in sickle-cell hemoglobin, by valine (see Plate 21). Here, conclusively, was evidence that genetic mutations – differences in the sequence of As, Ts, Gs, and Cs in the DNA code of a gene – could be "mapped" directly to differences in the amino acid sequences of proteins. Proteins are life's active molecules: they form the enzymes that catalyze biochemical reactions, and they also provide the body's major structural components, like keratin, of which skin, hair, and nails are composed. And so the way DNA exerts its controlling magic over cells, over development, over life as a whole, is through proteins.
But how is the information encoded in DNA – a molecular string of nucleotides, As, Ts, Gs, and Cs – converted into a protein – a string of amino acids?
Shortly after Francis Crick and I published our account of the double helix, we began to hear from the well-known Russian-born theoretical physicist George Gamow. His letters – invariably handwritten and embellished with cartoons and other squiggles, some quite relevant, others less so – were always signed simply "Geo" (pronounced "Jo," as we would later discover). He'd become interested in DNA and, even before Ingram had conclusively demonstrated the connection between the DNA base sequence and the amino acid sequence of proteins, in the relationship between DNA and protein. Sensing that biology was at last becoming an exact science, Gamow foresaw a time when every organism could be described genetically by a very long number represented exclusively by the numerals 1, 2, 3, and 4, each one standing for one of the bases, A, T, G, and C. At first, we took him for a buffoon; we ignored his first letter. A few months later, however, when Crick met him in New York City, the magnitude of his gifts became clear and we promptly welcomed him aboard the DNA bandwagon as one of its earliest recruits.
Gamow had come to the United States in 1934 to escape the engulfing tyranny of Stalin's Soviet Union. In a 1948 paper, he explained the abundance of different chemical elements present throughout the universe in relation to thermonuclear processes that had taken place in the early phases of the Big Bang. The research, having been carried out by Gamow and his graduate student Ralph Alpher, would have been published with the byline of "Alpher and Gamow" had Gamow not decided to include as well the name of his friend Hans Bethe, an eminently talented physicist to be sure, but one who had contributed nothing to the study. It delighted the inveterate prankster Gamow that the paper appeared attributed to "Alpher, Bethe, and Gamow," no less than that its publication date was, fortuitously, April 1. To this day, cosmologists still refer to it as the a(3y (Alpha-Beta-Gamma) paper.
By the time I first met Gamow in 1954, he had already devised a formal scheme in which he proposed that overlapping triplets of DNA bases served to specify certain amino acids. Underlying his theory was a belief that there existed on the surface of each base pair a cavity that was complementary in shape to part of the surface of one of the amino acids. I told Gamow I was skeptical: DNA could not be the direct template along which amino acids arranged themselves before being connected into polypeptide chains, as lengths of linked amino acids are called. Being a physicist, Gamow had not, I supposed, read the scientific papers refuting the notion that protein synthesis occurs where DNA is located – in the nucleus. In fact, it had been observed that the removal of the nucleus from a cell has no immediate effect on the rate at which proteins are made. Today we know that amino acids are actually assembled into proteins in ribosomes, small cellular particles containing a second form of nucleic acid called RNA.
RNA's exact role in life's biochemical puzzle was unclear at that time. In some viruses, like tobacco mosaic virus, it seemed to play a role similar to DNA in other species, encoding the proteins specific to that organism. And in cells, RNA had to be involved somehow in protein synthesis, since cells that made lots of proteins were always RNA-rich. Even before we found the double helix, I thought it likely that the genetic information in chromosomal DNA was used to make RNA chains of complementary sequences. These RNA chains might in turn serve as the templates that specified the order of amino acids in their respective proteins. If so, RNA was thus an intermediate between DNA and protein. Francis Crick would later refer to this DNA → RNA → protein flow of information as the "central dogma." The view soon gained support with the discovery in 1959 of the enzyme RNA polymerase. In virtually all cells, it catalyzes the production of single-stranded RNA chains from double-stranded DNA templates.
It appeared the essential clues to the process by which proteins are made would come from further studies of RNA, not DNA. To advance the cause of "cracking the code" – deciphering that elusive relationship between DNA sequence and the amino acid sequence of proteins – Gamow and I formed the RNA Tie Club. Its members would be limited to twenty, one for each of the twenty different amino acids. Gamow designed a club necktie and commissioned the production of the amino-acid-specific tiepins. These were badges of office, each bearing the standardized three-letter abbreviation of an amino acid, the one the member wearing the pin was responsible for studying. I had PRO for proline and Gamow had ALA for alanine. In an era when tiepins with letters usually advertised one's initials, Gamow took pleasure in confusing people with his ALA pin. His joke backfired when a sharp-eyed hotel clerk refused to honor his check, noting that the name printed on the check bore no relation to the initials on the gentleman's jewelry
.
The fact that most of the scientists interested in the coding problem at that time could be squeezed into the club's membership of twenty showed how small the DNA-RNA world was. Gamow easily found room for a nonbiologist friend, the physicist Edward Teller (LEU – leucine), while I inducted Richard Feynman (GLY – glycine), the extraordinarily imaginative Caltech physicist who, when momentarily frustrated in his exploration of inner atomic forces, often visited me in the biology building where I was then working.
Dna: The Secret of Life Page 8
