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36 THE LIFE SCIENCES out the history of biology, far-reaching general principles have been deduced by comparative study of related structures or functions across a variety of species, genera, or phyla. THE LANGUAGE OF LIFE The last two decades have witnessed a prodigious gain in understanding of life at all levels. Undoubtedly, however, the crowning achievement of this era has been the spectacular growth of understanding of that process central to life itself-the chemical encoding of genetic information, the mechanism whereby it is read out to give direction to the life of the cell, and the mechanism whereby it is reproduced in the course of cell division. This area of understanding variously termed molecular biology, bio- chemical genetics, the chemistry of reproduction, or the biochemistry of nucleic acids and proteins-flowered when the stage had been set. It could not have happened earlier and probably was inevitable when it did occur because of the centrality of the questions at issue. Until 1940, genetics had been studied, in the main, with conventional higher species of plants and animals, the hereditary traits studied being those most readily observed eye color, distribution of hair, flower pig- ments, etc. Through such studies, much of the language of formal genetics was generated, although the molecular mechanisms responsible were un- known. Biochemists had been concerned principally with identification and characterization of the chemical compounds characteristic of living forms and the pathways by which they are synthesized or degraded in cells. Microbiologists, long concerned with techniques for the identification and taxonomic classification of micro-organisms, then studied their susceptibility to sulfonamides and antibiotics as an adjunct to medical practice. Viruses had been a subject of study largely because of the diseases they engender in plant and animal species. By the mid-1940's it was possible to combine the understanding generated by these seemingly disparate disciplines into a concerted effort to understand the nature of the genetic apparatus. It had long been apparent that the genetic complement of any individual must be encoded in some chemical form; and, although morphological traits had served the geneticist well, in fact, there can be no gene for height, or eye color, or number of teeth, or age of onset of baldness. Clearly, the genes that govern such parameters must actually govern specific chemical events, the consequences of which are evident in these more readily dis- cerned characteristics. Slowly, the concept grew that each individual is a reflection of his complement of proteins; the latter serve both as structural

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FRONTIERS OF BIOLOGY materials and as the enzymes that synthesize all other types of biological chemicals. Each cell is whatever its proteins make possible, and the num- bers of cells of each type and the manner in which they are distributed are, in some way, a consequence of genetic instructions with respect to which proteins to make and the relative amounts of each. Belief in this concept began with the observations of Garrod in 1908, who assembled then-existing information concerning six hereditary diseases of man, indicating that each was the consequence of loss of some enzymic ability. This concept was solidified with studies of a bread mold, Neuro- spora crassa, which ordinarily can be grown on extremely simple nutritional media and synthesizes for itself all the usual amino acids, carbohydrates, purines, pyrimidines, vitamins, etc. Irradiated cultures of this organism were found to contain mutants that had lost the ability to make one or another of these vital components. By appropriate procedures it was ascertained which step in the sequence of chemical reactions by which such synthesis normally occurs (a "metabolic pathway" such as those shown in Figure 3) was actually blocked. In each instance, the mutant had lost the ability to catalyze one specific step in such a sequence. A large body of information accumulated from studies of a variety of bacterial forms confirmed this concept, encapsulated in the axiom, "A single gene deter- mines the synthesis of a single enzyme." The general relationship between the structure of genetic material and that of proteins, however, could not be established until the structural plan of proteins themselves had been revealed. The first protein to be studied appropriately was the pancreatic hormone, insulin, the structure of which is shown in Figure 4. Insulin proved to be constructed by the head-to-tail combination of 20 different kinds of amino acids; in all molecules of insulin, at each position along the chain, one and only one of the 20 possible amino acids does in fact exist. When the techniques developed for determination of this linear sequence became generally available, they were quickly applied to other proteins with similar results. Although a few differences were found among the insulins obtained from various species, in each species all the molecules of insulin are identical, as are all the molecules of cytochrome c, of myoglobin, and so on. These findings made explicit the nature of the information that must be encoded within the genetic material, i.e., instructions with respect to the linear sequence of amino acids in each of the proteins to be synthesized. If this concept. is correct, the amino acid sequence of a given protein must, in some manner, be colinear with the instructions within the gene responsible for its synthesis. The special advantage of utilizing micro-organisms is that it is possible to screen for mutants in populations of billions of individuals at one time and, by applying to them appropriate modifications of the classical tech

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38 THE LIFE SCIENCES

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FRONTIERS OF BIOLOGY 39 U. ~ ~ o ~ o Ct c ~ fi ~ ~ ~ Ct v o ~ . ~ ~ ~ o o ~ C ~ 3~ C; D C) ~ _] ~ ~ ~ _ . ~ ~ ~ Ct ~ ~ 04 _ ~ ~ , so O ^-S C) ~ ~ O O ~ ~ ~ ~ _ 3 'A ~ ~ V' =: ~ . _ ~ C~ > o Ct ~ ~ _ 0,, _ o C) ~ ~ ~ Ct _ ~ 9 L' _ - 5 5_ Cal ._ ._ ~ ~0 ._ ._ ~ ~ 3 o ~ ~ ~ o o ~ - 9 ~ O P: V, ha ~ ~ (O Ct ~ ~ a - C., a: Cal ~ ~ Cal ~ ~ O 3 .= "C Ct ~ Z ~ Ct C,= .o Ct ~ ~ ~V ~ O ' ~ O O s~ - O ~ ~ . ~ 9 `,L1 ~ ~ ~ p~ ~ ~ O C~) .C) ~ ^ O ~ .= 3 ~1 0 - Ct 1 P~ o C) ~n ~o o o C~ ~ _ . _ ~ 3 z ~i C~ _ cn r~ ~ _ ~ V) .C4

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40 THE LIFE SCIENCES HE ~ S S ~ ~H2 ~H2 ~H2 Gly Ileu Val Glu Glu Cy Cy Ala Ser Val Cy Ser I`eu Tyr Glu I`eu Glu Asp Tyr.Cy Asp 1 23 4 5 6 1 7 8 9 10 11 12 13 14 15 16 17 18 19 120 21 S S 1 1 NH NH S S 1 21 2 1 1 Phe Val Asp Glu His Leu. Cy Gly Ser. lIis Leu Val Glu Ala. Leu Tyr Leu Val Cy Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Ala 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 FIGURE 4 The amino acid sequence of bovine insulin. (From Principles of Bio- chemistry, 4th ea., A. White, P. Handler, and E. L. Smith. Copyright (I) 1968 McGraw-Hill, Inc. Used with permission of McGraw-lIill Book Company.) niques worked out by geneticists for larger organisms, construct "maps" of the genes. The earliest such maps, of the genes of fruit flies, simply related the position of each gene to that of other genes along a chromosome. The refinements possible with bacterial genetics enabled construction of de- tailed maps indicating the relative positions of individual mutations along the length of a single gene. Such techniques have been applied to a con- siderable variety of bacterial and viral genomes (the totality of genetic material in a cell). A rather thoroughly studied gene in this regard is that which directs the synthesis of an enzyme in E. cold called "tryptophan synthetase." The positions along the map of hundreds of mutants of this gene have been scrutinized and related to the accompanying change in the amino acid sequence of the protein itself. A partial summary of these studies is shown in Figure 5, which illustrates the colinearity of the gene that provides the instructions for making tryptophan synthetase and the amino acid sequence of that protein itself. B51 A38 A3 A33 A487 A23 A46 A7 8 A5 8 A96 A446 A223 A187 A169 Genetic map l l l l (not to scale) - =r I I I I =Jja Genetic map distances W.4~.7+0~1.6~.04~.3+.4~.0014.06~.55.001~.02~.3 - Amino acid in wild-type protein 1 Amino acid in m uta nt prot2 in I Position of change H2N-1-48 48 174 - 176-182 - 210 - 210 - 212 - 233-233 - 234 267-COOH In the protein , I :48+041 26~2+6~28+0+2~21~0+1-33 - Glu Glu Tyr Leu Thr Gly Gly Gly Gly Gly Ser Val Met Cys Arg He Arg Glu Val Cys Asp Leu Residue distance in polypeptide chain FIGURE 5 Colinearity of the amino acid sequence of the enzyme tryptophan synthetase and the substructure of the gene that governs its synthesis. ( From C. Yanofsky, G. R. Drapeau, I. R. Guest, and B. C. Carlton, '`The Complete Amino Acid Sequence of the Tryptophan Synthetase A Protein (car subunit) and Its Colinear Relationship with the Genetic Map of the A Gene," Proc. Natl. A cad. Sci. U.S. 57:296, 1967.)

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FRONTIERS OF BIOLOGY Figure 5 also reveals a concept first made clear by an understanding of the defect in sickle cell anemia. This disorder is the consequence of an alteration in the structure of hemoglobin, in which, at only one specific position (,86) along a chain of 146 amino acid residues, there occurs a substitution of the amino acid valine for glutamic acid. This observation, with its profound implications for the understanding of genetic disease, reveals the nature of the simplest possible kind of mutation: A change in the structure of the genetic material at one point along the strand of genetic instruction results in substitution of one amino acid for another in the strand of amino acids. The Genetic Material Meanwhile, an ever-growing body of evidence indicated that the genetic material itself must be the polymer called deoxyribonucleic acid (DNA), which is peculiar to the cell nucleus and is the stuff of which chromosomes are made. The ultraviolet-absorption maximum of this material occurs at the wavelength most effective in creating mutants. As techniques for the purification of viruses accumulated, each in turn was found to contain a nucleic acid as a major component. The capstone in this argument, which was not truly recognized as such at the time it occurred, was a study under- taken for rather practical clinical purposes. Two strains of Pneumococci were known, one of which was characterized by an outer coat of a carbo- hydrate polymer that the other strain lacked. When, however, cell-free preparations of cultures of the former were added to cultures of the latter, they were "transformed," acquiring the ability to make the carbohydrate polymer and retaining that ability through an indefinite number of subse- quent cell divisions. In effect, these cells had acquired a gene they formerly lacked. The material in the cell-free culture that made this possible was found to be DNA-in retrospect, categorical evidence that DNA is indeed the material of which genes are made. The lack of immediate appreciation of the profound implications of that finding was a consequence of earlier studies of the structure of DNA, which were misleading in that they sug- gested it was a dull repetition of a fundamental repeating polymer unit without variation. In light of such studies it had seemed unlikely that the structure of DNA could be the basis for genetic instruction, which was known to require immense variation. Only as the structure of DNA was re-examined was this incorrect impression rectified (Figure 6~. More careful analyses, using newer techniques, demonstrated great variability even in the gross structure, the relative composition varying from species to species, yet with one pair of cardinal rules. All DNA's

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42 THE LIFE SCIENCES o O=P-OH o / O \ H H ~ 13, 12' o l O=P-OH 41' H H Adenine H~C ~ O Guanine it,,: 41' H i, H 13, o l O=P-OH O=/-OH l H O Thymine -11' :/H FIGURE 6 A segment of the backbone structure of a single strand of DNA. Representation of a portion of a DNA chain, showing the position of the inter- nucleotide linkage between C-3' and C-5'. (From Principles of Biochemistry, 4th ea., A. White. P. Handler, and E. L. Smith. Copyright (if) 1968 Mc- Graw-Hill, Inc. Used with permission of McGraw- Hill Book Company.) are constructed of only four fundamental units called nucleotides: adenylic acid (A), guanylic acid (G), thymidylic acid (T), and cytidylic acid (C). This small number of units, nevertheless, permits encoding of a vast amount of information; indeed, in Morse code, with only two symbols, it is possible to transmit all the works of Shakespeare. In all specimens of DNA, A - T and G - C, whereas there is no consistent relationship between A and G. The meaning of this constancy was not apparent until combination of this information with studies of the x-ray-diffraction pattern of nucleic acids led to the now well-known depiction of DNA as two very long strands wrapped about each other in the familiar double helix, and so aligned that, on the strands, every A is opposed by a T. and each G is opposed by a C (Figure 7~. In each case the pair of bases is linked by the relatively weak forces of hydrogen bonds, as illustrated in Figure 8. The great stability of the double helix is the consequence of the sum of thousands of such unions. Importantly, there is no rule with respect to the actual consecutive sequence along one strand. If one knows the sequence

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FRONTIERS OF BIOLOGY 43 along one strand, one automatically knows the complementary sequence along the opposing strand, as shown in Figure 7. This structure immediately solved the two basic questions concerning the chemical structure of genetic material. This material must, as an in- trinsic property, both achieve its own self-duplication as cells divide and provide for the great variability that would permit the encoding of instruc- tions to make the enormous diversity of proteins found in nature. Self- duplication is achieved in a most ingenious way. Decades of bafflement concerning how any chemical could achieve its own self-duplication were resolved by the recognution that as cells divide, the double strand is, in some manner, disengaged and each strand then governs the synthesis not of itself but of its complement. Where one double strand existed, two double strands are brought into being, each of which contains one of the original strands and a complementary new partner (Figure 91. A great body of evidence now supports that concept, although some of the details remain obscure. The base pairing described is the feature of the structure that determines how the new strands shall be formed. Occasionally mis- takes are made, and when this occurs there is poor fitting of the strands. Under those circumstances a set of additional enzymes comes into play. One snips out the ill-fitting sections, thereby permitting another to reinsert / ~ . . . `~ .~ i ,~ 1 To 4~.\ f 1- At 13.4 A ~ A FIGURE 7 Schematic representation of the double helix of DNA. The ribbons represent the deoxyribosephosphate backbone chains. The op- posing arrows indicate that one strand is running from the 5' position of one sugar to the 3' posi- tion of the next, while the other strand is running in the opposite sense. The horizontal lines repre- sent hydrogen bonds between opposing pairs, two for each AT couple, three for each GC couple. (From I. Herskowitz, Genetics, 2nd ea., 1962. Copyright (I) 1962 Little, Brown and Company.)

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44 THE LIFE SCIENCES cytosi ne ~3 Th ~ ~ ~ ~ --in ~ -POSSE \ \ .\ 51.5] hi\ iTo chain To chain = ~_ A \ - - /~'. ~ 1 1 ~, ~ . ~. - 10 add (id) ~ To chain \ ~- - \ - - - - - (b) - / FIGURE 8 Molecular dimensions and hydrogen bonding of base pairs of DNA. (Adapted from S. Arnott, M. H. F. Wilkins, L. D. Hamilton, and R. Langridge, "Fourier Synthesis Studies of Lithium DNA, Part III, Hoogsteen Models," J. Mol. Biol., 11:391~02, 1965, p. 392.)

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FRONTIERS OF BIOLOGY 45 I_ ~L I_ _ (~ ~ ---I \ it- _ ~ \ _ _ fit 1: >~-----iLK 1 ~, , _ ~An_ ~ As_ 1 > - E-----5K I___ _ FIGURE 9 Scheme of replication of a DNA model. Boldface chains represent the newly synthesized strands of the two daughter molecules. (From J. posse, A. D. Kaiser, and A. Kornberg, "Enzymatic Synthesis of Deoxyribonucleic Acid," J. Biol. Chem., 236:864, 1961. Copyright (A 1961 The American Society of Biological Chemists, Inc.) the proper bases, which then make the normal tight fit of the double helix. Interestingly, the double helix of a bacterial chromosome, like that of most viruses, is a circular molecule, the head, as it were, being joined to the tail. This was originally recognized by the mapping procedures noted above, and then it was visualized by electron microscopy. Duplication of the DNA, therefore, must commence by opening this circle. In this concept, instructions for protein synthesis must be provided by the linear sequence of bases along the DNA strand; given the fact that the chromosome of E. cold consists of a single helix of about 10 million con- secutive base pairs, and that any one of the four bases may lie to left or to right of any other base, there is essentially an unlimited number of statistical possibilities, only one of which is the actual structure of a specific DNA chromosome. And the possibilities become astronomical in a human cell, the DNA complement of which is 1,000 times as great as that of E. coli. Subsequent research efforts concentrated on the mechanisms by which the ~ :,:`, (~1 _____

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46 THE LIFE SCIENCES instructions encoded in the DNA are utilized to give direction to protein synthesis. This is now understood in remarkable detail, although there are a few serious gaps in this knowledge. The summary that follows encom- passes the work of hundreds of investigators over a period of two decades. PROTEIN SYNTHESIS At an early stage it was recognized that, although the DNA holds the primary instructions for protein synthesis, it does not itself directly par- ticipate in that process. In the cells of higher organisms, DNA is locked in the nucleus, whereas protein synthesis occurs in small bodies called `'ribosomes" stippled throughout the cytoplasm. It followed, therefore, that the instructions for protein synthesis in the DNA must be dispatched from the cell nucleus to the ribosomes. Ribosomes were found to be com- posed of a mixture of about 20 different proteins plus several forms of ribonucleic acid (rRNA); RNA differs from DNA in that the sugar com- ponent is ribose rather than deoxyribose, and most RNA is single-stranded rather than double-stranded. Ribosomes are leaflets constructed of a small component and a larger component, disposed much as a partially open clam. The messages from nucleus to ribosome were shown to consist of yet another form of single-stranded ribonucleic acid, messenger RNA (mRNA), which is fabricated in the nucleus by a specific enzyme called the DNA-dependent RNA synthetase. This form of nucleic acid is tran- scribed from one strand of the DNA helix by the same base-pairing rules, except that the pyrimidine nucleotide uridylic acid (U) is utilized, rather than the thymidylic acid (T) of DNA, so the four letters of the RNA alphabet are A, G. U. and C. Just as in DNA itself, the growing mRNA is formed on one of the DNA strands in an antiparallel manner (see Figure 91. In a living cell the long fibers of mRNA can be seen threaded through as many as 10 ribosomes at once (a polysome), so different areas of the message are being "read" by each of the ribosomes consecutively. Assuming some kind of colinearity of the mRNA and the protein to be synthesized, it must be the base sequence of the mRNA that specifies the amino acid sequence of the protein. Since there are 20 different amino acids in proteins and only four letters in the RNA alphabet (A, G. U. C), obviously these cannot bear a one-to-one correspondence; moreover, one can form only 12 two-letter words with four letters. Hence, the minimal number of letters that would suffice is three per "word," i.e., the "codon" that specifies the exact amino acid next to be incorporated in a growing protein chain. Indeed, one can form 64 three-letter words with a four- letter alphabet. The problem then, was to establish the relationship be- tween the four-letter alphabet of the RNA and the 20 words in the amino acid dictionary.

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FRONTIERS OF BIOLOGY If three-letter words are utilized and a large body of evidence now indicates that the code words for amino acids are built of three letters each there seemed no way physically to relate the structure of the amino acids to a sequence of three nucleotides in RNA. Accordingly, it was postulated that some form of "adapter" would be required. That adapter proved to be yet a third general type of RNA, termed "transfer RNA" (tRNA), the smallest kind of RNA known. Several pure tRNA's have been isolated, each of which serves as the adapter for one specific amino acid; Figure 10 shows a complete structure of one tRNA. All tRNA's appear to be built along the same general plan. Within the cell there is a family of "amino acid-activating enzymes," and the fact that the entire apparatus actually works successfully rests on the remarkable properties of these enzymes. In absolutely specific fashion, each such enzyme esterifies one and only one of the 20 amino acids to the hydroxyl group at the 2-position of the ribose at one end of one specific form of tRNA; it is imperative that the enzyme make no error since any such error would necessarily become an error in protein synthesis. What is shown in Figure 10 as the projecting round knob of the tRNA molecule is the "anticodon," a sequence of three bases which fit, by the usual base- pairing rules, against three consecutive bases in the messenger RNA, the codon for an amino acid. The amino acid is attached to the tRNA at a position quite remote from the anticodon. In a cell engaging in protein synthesis, there is a pool of all 20 amino acids, each affixed to its specific tRNA by virtue of the activity of the appropriate activating enzymes. As the long mRNA (500 to 10,000 nucleotide units) threads through the ribosome leaflet, it is attached to the smaller ribosomal component, while an amino acylated tRNA, which can achieve the necessary complementary base pairing to the message, is fixed in position on the larger member. The first amino acid to be laid down is that at the amino terminus of the chain. The protein chain grows by the reaction O R1 O Ret O 11 1 11 1 ~1 ------C- N CH C tRNA1 + HINT CH C tRNA., H O R1 O R 11 ~ 1 11 1 ------C N CH C N CH tRNA~ + tRNAl. H H As each such reaction is completed, the freed tRNA departs, and the mRNA must move through the ribosome so that the next three "letters" 47

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48 THE LIFE SCIENCES o 1 G) 1 1 G) ~ 1 1 G) 1 1 1 1 G) ~1 c 1 1 G) _ ~ _ ~ c _ :~ 9_ :~ _ c~ 1 1 1 1 , G- C- G- C - <;, am. \ c' / c \ 'G- O' o;~ v_9_9_~> _ >_ (~ - n ~ 1 1 1 1 1 u-c-C-G- G-r ~ c A)\ o-~ 1 1 C AS 1 1 ~V 1 1 1 1 l c 1 1 C _ ~ - I /- G _C I | ~ | Anticodon FIGURE 10 Base sequence and gen- eral structure of a tRNA for alanine. The anticodon, the three bases that pair with the three-base codon of mRNA, are shown at the bottom. As in DNA, and DNA-RNA hybrids the strand of tRNA is running in the op- posite sense to that of the mRNA, to which it must attach on the ribo- some surface. (From J. T. Madison and H. K. Kung, "Large Oligonucleo- tides Isolated from Yeast Tyrosine Transfer Ribonucleic Acid after Par- tial Digestion with Ribonuclease T1," J. Biol. Chem., 242: 1324-13 3 O. March, 1967, p. 1329. Copyright (it) 1967 by The American Society of Biological Chemists, Inc. ) are aligned at the working site. The mechanism by which this ratchet-like process is accomplished is totally unknown. These events are schematically shown in Figure 11. There remained the task of establishing the dictionary; as the decade of the 1960's began this appeared to be a herculean task. A fortunate combination of accident and experimental virtuosity rapidly broke through this problem, the solution to which is shown in Figure 12. All 64 pos- sible three-letter words are utilized and, hence, show considerable redun- dancy. Where more than one code word is utilized to signify the insertion of a given amino acid, an equal number of tRNA's must also be available

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FRONTIERS OF BIOLOGY to the cell; in several instances this has been shown to be the case. As will be seen, three of the three-letter words do not relate to any amino acid; where these occur in the mRNA sequence, no amino acid can be inserted, and synthesis of the protein chain terminates and the tRNA at the end of the cliain is removed by hydrolysis. This, therefore, is the "punctuation" in the message, the "period that ends the sentence." Understanding of exactly how chain initiation is accomplished is less satisfactory. It is all-important that reading of the message begin at a pre- cise point; if the reading frame were to shift by one letter, the entire message would be garbled and a completely different set of amino acids would be assembled. In bacterial forms, it would appear that the first amino acid in the chain is always the same-methionine, the amino group of which bears a formyl group. Message reading, therefore, begins by utilizing formyl methionine tRNA as the first "word" and is terminated when any one of the three nonsense words shows in the message. Acetyl serine tRNA may serve the same role in animal systems. One of the most powerful tools in the multitude of studies that gave rise to the remarkable picture just described was the experimental demonstration of the effectiveness of base pairing. The force that holds the two strands of DNA together is the sum of a multitude of tiny forces hydrogen bonds between nitrogen and oxygen atoms. These are the forces that also hold Formyl-Met-Phe-Lys~ /Pro 5/\ A U G U 1 1 1 1 into of\ 1 \~\ ~G U U A A A C C \C U 1 1 1 1 1 1 1 ~1 I yr~ Ala -~ ~1 ~ ' Tyr tRNA Ala tRNA 1 1 1 C G I 1 1 1 1 1 1 /~ / 1 1 1 1 1 1 1 1 1 1 1 A C/ G C C / U C C 1 J<% 1 1 ~ 1 1 1 1 1 - _' ~- ; ~mRNA1 (50 S) Ribosome (30 S) Movement of ribosomes FIGURE 11 Schematic representation of protein synthesis on a ribosome. The pro- tein tRNA is departing, having been used in the previous step; the bond between tyrosine and its tRNA is being displaced by the amino group of alanine; and a seryl- tRNA is just coming into position. (From Principles in Biochemistry, 4th ea., A. White, P. Handler, and E. L. Smith. Copyright (I) 1968 McGraw-lIill, Inc. Used with permission of McGraw-IIill Book Company.)

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50 THE LIFE SCIENCES z o - E~ V) o Pi - 2nd POSITION U C A G PHE SER TYR CYS PHE SER TYR CYS U LEU SER CT- 1 CT-3 LEU SER CT-2 TRY LEU PRO HIS ARG LEU PRO HIS ARG C LEU PRO G LN ARG LEU PRO GLN ARG ILU THR ASN SER A ILU THR ASN SER ILU THR LYS ARG MET THR LYS ARG VAL ALA ASP GLY VAL ALA ASP GLY G VAL ALA GLU GLY I VAL ALA GLU GLY U C A - U C A - U C A G U C A G z o - v, o FIGURED The genetic code. Each amino acid in a protein is specified by a nu- cleotide triplet in RNA, e.g., aspartic acid (asp) is specified by the triplets GAU and GAC. UAA,UAG, and UGA are utilized for punctuation, viz., to indicate when to terminate the chain. crystals of water, i.e., ice, together and, like ice crystals, they can be melted by warming. If double-stranded DNA is brought to an elevated temperature, the double helix comes apart and the DNA becomes indi- vidual random flopping coils. If the temperature is then lowered very slowly, the coils find each other and the double helix is restored. By the same technique one can prepare in the laboratory hybrid DNA-RNA complexes, but only-when the latter can be aligned and can join by multitudinous base pairing. In this way it was shown that a rather substantial fraction of DNA of E. cold and a much larger fraction of mammalian DNA are used to code for the preparation of rRNA, but only a tiny fraction, less than 0.1 percent, specifies the formation of all the tRNA's. And by the same procedure one can artificially achieve attachment of tRNA onto RNA. Indeed, this tech- nique was the basis for the most successful procedure for determination of the genetic code. Thus? a trinucleotide of RNA of known base sequence can be added to a ribosome suspension. To a sample thereof is added a tRNA charged with its amino acid, the latter labeled with ~4C. If the

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FRONTIERS OF BIOLOGY radioactivity adheres to the ribosome-RNA complex, the tRNA has paired its anticodon with the trinucleotide and the code word is established. Much of these concepts had been elaborated by deduction from a great variety of experimental observation. A capstone on this intellectual struc- ture was provided by a series of maneuvers, conducted with great technical skill, which achieved the isolation, in pure form, of a single gene from among the many of the genome of E. colt, that which directs the synthesis of any enzyme called 3-galactosidase. The final proof of this overall picture has been provided by a remarkable tour de force. From knowledge of the struc- ture of the tRNA for alanine, an antiparallel, complementary length of DNA was synthesized chemically. The usual DNA-synthesizing enzyme was utilized to form its complementary DNA strand. This relatively short double-stranded DNA was then used with the DNA-dependent RNA- synthesizing enzyme to form the tRNA for the amino acid alanine and yielded the predicted structure. This constitutes true chemical synthesis of a gene! Similarly, synthetic lengths of RNA have been used with ribosomes as mRNA and have yielded small polypeptides of the structure predicted by the code. Self-Assembly Such studies demonstrated that base pairing is the primary mechanism involved in DNA duplication, in the synthesis of RNA on DNA, and in message reading in the ribosome. But they also demonstrated a cardinal principle of the biological world, the principle of self-assembly. Organisms are assemblages of cells, and within the cells there are myriad organized sub-cellular bodies, within which, in turn, are macromolecules that are aggregates of smaller molecules. Yet, as we have seen, all that genetic instructions can provide is information descriptive of the synthesis of protein chains. Are other types of instructions required, or does all else follow from the fact of protein-strand synthesis? The answer, unques- tionably, is that all else is derivative, that all other structures combine, because they do indeed fit together and are held together by a collection of small forces, much as are the two strands of DNA that make such a remarkably tight fit. For example, the hemoglobin molecule consists of four subunits, two a-chains, and two -chains. The two chain types can be separated by appropriate means but, when remixed, the normal tetrameric units recon- stitute themselves without assistance. The enzyme ribonuclease is a single protein chain within which are three internal disulfide bridges ~ S S-). These can be opened by appropriate chemical means (reduction). In this form the enzyme is a random flopping coil and lacks catalytic activity. When allowed to reoxidize slowly, virtually every molecule regains its enzymic activity, despite the fact that there are eight different ways in which the