THE CHEMISTRY of Life - C&EN Global Enterprise (ACS Publications)

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Chemisty

Chemistry

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This week, CirEN begins a three-part series on the chemistry of life. First, we present this special report by the C&EN staff on how living cells synthesize proteins. Next week, Dr. Heinz Fraenkel-Conrat and Dr. Wendell M. Stanley tell what they and others are learning about the RNA viruses and how their findings increase our understanding of how genetic information is transmitted. Finally, in the third article, Dr. Melvin Calvin will discuss our present knowledge of the origin of life on earth.

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chemical science series

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Jiow Cells Synthesize Proteins Scientists make steady progress on this basic mechanism, find it's tied to the cell's nucleic acids

The central problem of biochemistry today is how living cells synthesize protein. In the burgeoning field of nucleic acid research, probably more investigators are working on this complex process than on any other. Biochemists, physical and organic chemists, and geneticists are among the many who have taken up the challenge. Prime purpose of this growing army of scientists is to shed light on the complex route from amino acids to protein. Most of the route is still very murky, and much of it is dark. Not only are proteins the underlying structure of all living organisms, but they are the major part of the organisms' enzymes, antibodies, and many hormones. That proteins make

THE KEY PROBLEM. The amino acid sequence of ribonuclease and a micrograph (magnification 35,000 X) of part of a pancreatic cell symbolize the problem: How do cells manage to build these highly specific structures?

up the machinery of living things ;s should alone be enough to explain thee widespread activity in this area. Butit there is another, and inseparable, facet;t that intrigues many scientists evenn more. Protein synthesis is the major»r path for putting genetic information too work. 's Through protein synthesis, the genes enforce their inherited law; they buildd cells, organs, and organisms, then govern their metabolism. The full pic> ture, many workers believe, will showv an unbroken chain of events leading g to deoxyribonucleic acid ( D N A ) . This is material carries all the hereditaryy traits of a species—and of an individualtl —•coded in the nucleotide sequencee talong its length. One of the most important hereditary traits is the aminoo acid sequence of every single proteinn needed by the species. e It's no coincidence, then, that the >greatest advances in knowledge of protein synthesis have come during thee same period as those in nucleic acid d chemistry. The two fields interweavee icontinually, and they may be the ulti-

mate meeting ground of chemistry and genetics. Twenty years ago chemists thought of protein synthesis simply as the reverse of protein breakdown. Another popular idea was that a protein was synthesized all at once from free amino acids by some sort of template medianism. The past decade has shown how oversimplified these notions are. Dr. Mahlon B. Hoagland of Massachusetts General Hospital in Boston believes that the present upswing in interest exists because the intermediate reactions in the synthesis can now be dealt with as chemical problems. "Earlier, we struggled with bewilderingly complex systems in which great satisfaction was derived from finding that an amino acid really found its way into protein at all," he says. Now chemical equations, based on experimental data, appear in the literature on protein synthesis. Why is the mechanism of protein synthesis such a formidable problem? Scientists must find out how living cells use the same building blocks to put MAY 8, 1 9 6 1 C&EN

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together such different structures as the proteins of muscle, blood, milk, hair, enzymes, and so on. The prob­ lem goes even farther than how pep­ tide bonds are formed and how amino acids are arranged in specific se­ quences. Other important facets in­ clude: • What processes give protein chains their specific configurations and link­ ages? • How are proteins bound to low molecular weight compounds, such as heme, metals, coenzymes, and the like? Current knowledge divides the scheme into three major parts. Al­ though the divisions are largely arbi­ trary, chemists find them convenient for any discussion of the synthesis. The first—and best understood—is ac­ tivation of the amino acid. Next in line is linking the amino acid to a relatively small, soluble ribonucleic acid (sRNA). Then this intermediate, according to the theory, attaches it­ self to a specific spot on a large RNA template in cellular particles called microsomes. This is where amino acids—each on its own specific sRNA— link together to form a protein. The First Key As early as 1941, Dr. Fritz Lipmann of Rockefeller Institute suggested that the energy of a phosphate bond might activate the carboxyl group of amino acids, as it does in other biochemical processes. But more than a dozen years passed before this idea took con­ crete form. In 1953, Dr. Lipmann and his associates, working on a different problem, were studying the transfer of energy from adenosine triphosphate (ATP) to form acetylcoenzyme A. They discovered that the reaction liberated a pyrophosphate group (PP), instead of the expected phosphate group, and adenosine monophosphate (AMP): Acetate -f- ATP + CoA - * Acetyl-CoA - f AMP - f PP Dr. Paul Berg at Washington Uni­ versity (St. Louis) showed that the intermediate of the reaction is an acetyl adenylate. Further work demonstrated that the reaction isn't limited to activating the acetate car­ boxyl group; it works with other acids as well: RCOOH + ATP - * RCO-AMP + 82

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PP

Chemists sorely needed a general re­ action like this to explain the activa­ tion of a score of different amino acids involved in protein synthesis. Dr. Hoagland and his colleagues at Massa­ chusetts General, Dr. Paul Zamecnik and Dr. Ε. Β. Keller, took advantage of this breakthrough and soon con­ verted it into another one. In rat liver they found enzyme ac­ tivity for the reversible reaction be­ tween amino acids and ATP to give aminoacyl adenylates and pyrophos­ phate. They also showed that sepa­ rate enzymes, rather than separate sites on the same enzyme, catalyze the activation of the different amino acids. Similar observations were made by Dr. Berg and by Dr. J. A. DeMoss and Dr. G. D. Novelli using bacterial sys­ tems. And so the activation step took shape: Amino Acid -f- ATP -f- Enzyme (E) ^± aa-AMP-E + PP Ever since discovery of these en­ zymes, work has gone on steadily to separate them. Dr. Lipmann, with Dr. E. W. Davie and Dr. V. V. Koningsberger, isolated a partly purified preparation of the tryptophan-activating enzyme. At Tufts University School of Medicine, Dr. Marvin Karasek, Dr. Paul Castelfranco, and Dr. Alton Meister showed in a direct way that enzyme-bound aminoacyl adeny­ late actually forms in these systems. They reacted labeled tryptophan with ATP and an excess of tryptophan-activating enzyme, then isolated the tryptophanyl adenylate at the end of the reaction. Many enzymes from bacteria, birds, and mammals have been purified to various degrees. Some amino acids show little activity toward the en­ zymes. But this might be due to less stable bonds which don't survive the methods used to detect the complex. There seems to be little doubt, Dr. Hoagland says, that activating enzymes exist for each of the amino acids. Having catalyzed activation of the amino acids, the enzymes still have a second important job to do. The First Lock During the work on the activation step, no measurable amounts of free aminoacyl adenylates were found in solution. Evidence was strong that they exist. The mechanism fit, but the acceptor for the amino acid was

still missing. What, then, became of the aminoacyl adenylate complex with enzyme (aa-AMP-E)? It would have been convenient if the complex went directly to the microsome particles, attached itself to a specific spot on the RNA template, and completed the protein synthesis. But instead, investigators found signs of another route. Dr. T. Hultin and Dr. G. Beskow in Sweden and Dr. R. W. Holley at Cor­ nell University obtained indirect evi­ dence that some intermediate is in­ volved in the gap between amino acid activation and protein synthesis. Re­ search on the mechanism passed an­ other milestone when Dr. Hoagland, Dr. Zamecnik, and Dr. Mary Stephen­ son—and independently Dr. K. Ogata and associates in Japan—showed that the activated acids are transferred to soluble RNA (now also called sRNA, transfer RNA, or acceptor RNA by var­ ious workers). Soluble RNA is relatively small com­ pared to the giant nucleic acids in microsomes and cell nuclei. Estimates of sRNA's molecular weight vary from 25,000 to 35,000. This means only 75 to 90 nucleotides per chain. Chemists soon found that the amino acids form covalent links to sRNA and that the reaction is catalyzed by the same preparations that contain the activating enzymes. The nature of the reaction became clearer as information accumulated : • In intact cells, the amino acid link to sRNA occurs before amino acids show up in any other RNA or in pro­ tein, Dr. Hoagland and co-workers found. • Soluble RNA seems to be unique among nucleic acids in its ability to bind amino acids. RNA from micro­ somes, cell nuclei, and viruses ( as well as synthetic RNA) is inactive in this respect. • The same enzyme that activates an amino acid catalyzes its transfer to sRNA. This finding resulted from work by Dr. R. S. Schweet and co­ workers at the University of Kentucky and by Dr. E. J. Ofengand and Dr. Berg. • The terminal nucleotide sequence in sRNA is cytidylic-cytidylic-adenylic (or C-C-A, in the notation used by workers in the field). Dr. Zamecnik's group found that this arrangement at the end of the chain is necessary for amino acid binding to sRNA. If this

CLOSE-UP OF RNA, THE DIRECTING AGENT OF PROTEIN SYNTHESIS Adenine (A)

Guanine (G)

Cytosine (C)

Uracil (U)

Two purines and two pyrimidines are important parts of the RNA structure.

Attached to ribose, they make up the four ribonucleosides. ADENOSINE Putting — OPO3H on the 5'-carbon in place of the OH gives the structures of the four key ribonucleotides.

GUANOSINE

Adenylic acid Guanylic acid or adenosine monophosphate (AMP)

CYTIDINE

URIDINE

Cytidylic acid

Uridylic acid

RNA is a polyribonucleotide linked by phosphodiester bonds between the 3'and 5'-carbons of ribose. The nucleotide sequence for specific RNA's is unknown. sRNA has the same structure but a much smaller chain. Each chain ends with the sequence -C-C-A, but somewhere within the chain is a "code" sequence for the specific amino acid it can carry, shown here attached to the ribose of the terminal adenylate unit.

aa-sRNA

Coupling sRNA with template RNA may take place through specific hydrogen bonding —- A only with U and G only with C.

terminal grouping is removed, the sRNA can't act as an acceptor. • Dr. Lipmann's group—and independently, Dr. Zamecnik and co-workers—showed that the amino acid is attached to the terminal adenosine fragment of liver sRNA; specifically, it is linked to the 2'- or 3'-carbon of the ribose fragment in an ester linkage. Dr. Berg and his associates reached the same conclusions using soluble RNA from Escherichia coli. Which of the two possible carbons of the ribose is the actual site is still the subject of active research.

Up to this point, then, chemists could draw a partial structure for aminoacyl-sRNA. But there was still an important, unanswered question about sRNA. Is there a specific one for each amino acid? In the reaction with sRNA, many workers noted, several different amino acids, when used together, do not compete for binding sites. Instead, the effect is additive; each boosts the total activity toward sRNA, as if heading for its own specific site or its own specific sRNA. Many investigators have been able to enhance sRNA's ac-

tivity toward certain amino acids by purifying it partially. But the separation of sRNA into an array of specific sRNA's, or even the isolation of a single pure one, hasn't yet been achieved. Many research workers, however, are pursuing this cherished goal. Among them are Dr. Holley, Dr. Geoffrey Brown (Kings College, London), and Dr. Zamecnik's group. Despite the lack of pure compounds, the weight of evidence is on the side of a specific sRNA for each amino acid. This would explain why the MAY

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NEW PROTEIN. Electron micrograph (magnification 19,500 X) by Dr. Sanford L. Palay of the National Institutes of Health shows parts of four cells of the rat pancreas. At the point where they meet, there is an extracellular canal called the lumen (L) f containing masses of newly synthesized protein, zymogen (Z). Other zymogen droplets and two mitochondria (M)—the energy centers of the cells—can be seen in the cytoplasm of the cells. Striations of tiny dark parti­ cles throughout the cytoplasm mark the endoplasmic reticulum. When the cells are homogenized and centrifuged, the endoplasmic reticulum and the as­ sociated tiny particles yield microsomes, the site of protein synthesis

different amino acids don't compete with each other in the reaction. In the first two steps of protein syn­ thesis, a given amino acid (aa x ) may go through these reactions: (1) aa r - f ATP - f Enzyme ( E ^ — aarAMP-Ei - f PP (2) a a 1 - A M P - E 1 + sRNA! ^± aax-sRNAj - f AMP - f E±

Each of the amino acids involved in protein synthesis, according to these ideas, goes through these steps, each with its specific enzyme, each winding up attached covalently to a specific sRNA. The medium surrounding the microsome particles, then, is teeming with aa 1 -sRNA 1 , aa 2 -sRNA 2 , and so on. An activating enzyme in protein synthesis up to this point does two things: It catalyzes formation of aaAMP-E (efficiently rejecting the un­ natural D-amino acids); it also serves 84

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as the first key in what may turn out to be a series of lock and key steps leading to the genetic information in DNA. In this second role, the enzyme must recognize a specific sRNA that will accept the amino acid it has acti­ vated. But all the sRNA's, it turns out, have the same terminal group of nucleo­ tides, C-C-A. Thus, the enzyme must recognize some other feature of the polynucleotide, possibly a particular sequence farther along in the chain. Dr. Zamecnik and co-workers sug­ gest that some feature in addition to nucleotide sequence may distinguish the sRNA's. Their reasoning is based on the relative ease of getting partial separations of specific sRNA's from each other by purely physical means. The differences may lie in secondary or tertiary structure, such as hydrogen bonding and folding of the chain. Some large surface fit of sRNA to en­

zyme, they postulate, might be re­ sponsible for the recognition of one by the other. Ultimately, though, the nucleotide sequence of each sRNA de­ termines how the molecule folds into its own particular physical shape. How the key fits into the lock and how it operates is still completely un­ known. This is another big objective of current research. Chemistry

of Final Stage

The chemistry of the final stage of protein synthesis is obscure. Much of what is known here has come from Dr. Zamecnik, Dr. Hoagland, and their associates, Dr. Mary Stephenson, Dr. J. F. Scott, Dr. L. I. Hecht, and Dr. Ε. Β. Keller. They find that a system containing amino acid-sRNA, micro­ somes, and guanosine triphosphate (GTP) rapidly incorporates the amino acids into protein. In addition, the results show that the amino acid does not pass through a free state. It was then found that the later

stages also need ATP somewhere along the line. Dr. Hoagland speculates that the ATP may tend to push the activation-transfer reactions to the right. ATP +

aa +

sRNA ^± aa-sRNA - f AMP +

PP

In Dr. Lipmann's laboratory, Dr. W. C. Hiilsmann and Dr. George Acs also found that ATP and GTP are needed. Dr. Lipmann suggests that the relatively large amount of ATP required may mean another energy turnover at this stage, just as in the activation of amino acid at the beginning of the reaction scheme. More recent work by Dr. Kivie Moldave indicates that ATP is not necessary in the actual transfer (although possibly in another step). But there is some unknown factor (possibly an enzyme) that is needed. He has purified this to some extent and is trying to find out what it is. But at some time during synthesis, Dr. Hoagland finds, the sRNA and its amino acid become associated with the RNA of the microsomes. When aasRNA (labeled in the amino acid or in its pyrimidine bases) is incubated with GTP and microsomes, the labels show up in the high polymer RNA. This does not happen with labeled sRNA by itself. Thus, it appears that each sRNA ( or a section of it) with its amino acid finds its own particular site on the giant RNA template. This would line up the amino acids in the sequence dictated by the template. The code that sets the site for a particular aa-

sRNA on the template is unknown. But this scheme, which has two nucleic acids interacting with each other, suggests some strong possibilities. One widely held theory to account for the strict specificity of protein synthesis has its roots in nucleic acid structure. The Watson-Crick structure for DNA has two deoxyribose-phosphate chains wound around each other in a double helix. To each deoxyribose unit, a purine or pyrimidine is attached—adenine, thymine, guanine, or cytosine (A, T, G, C ) . The helix, according to the theory, is held together by hydrogen bonding between the purine and pyrimidine bases on the different chains. But adenine can hydrogen-bond only to thymine, and guanine only to cytosine. Thus, one chain is the complement of the other. Where A-T-G-G appears in one, T-A-C-C appears in the other. Natural RNA is generally believed to be single-stranded. (But there are indications that a large fraction of microsomal RNA is helical and shows base pairing.) Whatever RNA's secondary structure, its primary structure is similar to that of DNA. RNA is a ribose-phosphate polymer with a purine or pyrimidine base on each sugar unit. Three of the four bases are the same as in DNA (A, G, C ) ; the fourth is uracil, in place of thymine. Using synthetic polyribonucleotides, Dr. Alexander Rich and co-workers at Massachusetts Institute of Technology have shown that RNA can form twoand three-stranded complexes that also

use hydrogen bonding between purine and pyrimidine bases—adenine with uracil, and guanine with cytosine. Both DNA and RNA can code vast amounts of information in the sequence of base units along the huge chains. Every important and minor characteristic of a living organism is probably carried in this way in the nuclear DNA. Before the discovery of sRNA, most theories on the final stage of protein synthesis assumed that the template RNA must interact directly with the amino acids. Research on soluble or "transfer" RNA's, however, led Dr. Hoagland and his associates to propose another mechanism. Similar ideas had already been put forward on theoretical grounds by Dr. F. H. C. Crick of Cambridge University. He suggested that an "adaptor" molecule might help an amino acid find its

DNA DOUBLE HELIX. The WatsonCrick structure of DNA has two deoxyribose-phosphate chains wound around each other in a double helix. To each deoxyribose unit is attached a purine or pyrimidine (adenine, thymine, guanine, or cytosine). The helix, according to the theory, is held together by hydrogen bonding between the purine and pyrimidine bases on the different chains. Since adenine can hydrogen-bond only to thymine while guanine can hydrogen-bond only to cytosine, one chain complements the other. Where A-T-G-G appears on one, T-A-C-C appears on the other. The twisted band represents the sugarphosphate-sugar-phosphate chain. The arrows show the direction of the linkage:

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specific place on the template. The properties of the sRNA's are just what Dr. Crick's hypothesis dictates for the adaptor molecules, and so experiment and theory fitted together neatly. Since protein synthesis is remarkably specific, the mechanism must operate with extreme precision. Direct amino acid interaction with the template probably can't do this; specific sites on template RNA would have to be able to recognize the R groups of the different amino acids. But purine-pyrimidine interaction of two nucleotides can satisfy the demand for precision. If an amino acid is first attached to an sRNA that has the sequence A-C-U at some critical point in the chain, this group can pair only where there is a U-G-A sequence on microsomal RNA. And so, according to the hypothesis, the amino acid first reacts with this adaptor and assures its proper place on the template. At no time does the amino acid have to meet the template. A crucial factor in this mechanism is a code. Each sRNA must have a nucleotide grouping that is specific for one amino acid. And the complement of this code must exist on the template RNA. To code for 20 amino acids requires permutations of the four basic nucleotides, taken only three at a time. Thus, the triplet A-A-U might code for one amino acid, A-C-A for a second one, U-G-A for a third, and so on. Although three nucleotides are enough for a code, more may be involved in the actual coupling to template RNA. But sRNA has 75 to 90 nucleotides in its chain. If more than a few of these couple with template RNA, the amino acids at the ends of the chains may be too far apart to form peptide bonds. Chemists must learn much more about the arrangement of RNA in the microsomes before they can say just how this predicament is resolved in the particle. Dr. Hoagland and co-workers suggest that the shape of the RNA template chain may bring the amino acids together. One possibility is a flattened helix in which the sRNA "code" nucleotides pair with their complements along each turn of the helix, with part of each sRNA and its amino acid extending beyond the helix. The amino acids attached to the sRNA's would line up parallel to the screw axis. A similar scheme, suggested by Dr. Lipmann, puts the coding sequence at one end of sRNA, which is 86

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FACT AND THEORY COMBINE IN THIS OVER-ALL PICTURE Activating and Linking Amino Acid to sRNA

ADENOSINE TRIPHOSPHATE (ATP)

AMINO ACID

sRNA

Assembling the Protein Leaves template after its aa4 is attached to aa2

Finds place on template

Template RNA This hypothetical scheme shows one way amino acids might line up in a specific order in the later stages of protein synthesis. The theory presumes that a different "code" sequence of nucleotides is present on each sRNA. If the template RNA carries the complement of each code group, the order of these groups on the template can dictate the order in which the amino acids will hook up in peptide link ages. Whether such codes actually exist is still a matter of speculation. The sequences shown here were chosen only to illustrate the theory

attached to the template. The rest of the sRNA with the amino acid hangs from the template with enough freedom to bring the amino acids together. All these pictures, Dr. Lipmann emphasizes, shouldn't be taken as any more than "a very rough attempt to make some sense" of the chemistry of the final step. Other chemists, also, stress this point. Pertinent to the coding question are some results of Dr. Maxine Singer and Dr. Giulio Cantoni of the National Institutes of Health. Their work suggests that just as all sRNA's have a C-C-A on one end, they also have a terminal guanosine group on the other end. How this fits into the picture is not clear, but it does indicate that the code may not include the terminal guanosine. One result of the adaptor hypothesis is the prediction that adaptors and microsome particles should be interchangeable from species to species, since all living systems build their proteins from the same 20 amino acids. Species specificity, then, should exist in the order of the amino acids, not in the mechanism for achieving the order. This is what has been found. For activating enzymes, the situation is less clear. Bacterial enzymes either don't transfer amino acids to animal sRNA or do a relatively poor job of it. But bacterial sRNA already charged with amino acids is interchangeable with charged animal sRNA. Thus, the adaptor code seems to be universal, but the code on the activating enzyme apparently is not. The ultimate protein, though, must be characteristic of the species that donates the microsomes (containing the RNA templates). There are indications that this is so. Dr. Schweet's group has obtained evidence that a system containing rabbit reticulocytes and guinea pig liver soluble fractions forms hemoglobin, the expected product of reticulocytes. One jarring note comes from Dr. Saul Kit of the M. D. Anderson Hospital and Tumor Institute in Houston. Other workers, he points out, find that the ratios of G-C to A-T of bacterial DNA vary widely from species to species, but the bacterial RNA base ratios are much narrower. Mouse DNA base ratios obtained in Dr. Kit's lab fall in the middle of the bacterial range. But mouse RNA base ratios are completely out of the range of any bacterial RNA data. "This is very strange," he says, " if we assume

a universal code in which the nucleotide base sequence provides information from DNA to RNA to proteins." Dr. Kit's results have caused some concern. But research by Dr. Elliot Volkin and Dr. L. Astrachan at Oak Ridge National Laboratory and by others may point to a way out of the dilemma. Shortly after infection by a DNA-containing bacteriophage, a bacterial cell starts to make an RNA that is smaller than microsomal RNA, they find. This new RNA shows a base ratio that is similar to that of the phage's DNA. The results suggest that only a small part of microsomal RNA is template RNA. And so the base ratio of total RNA may not reflect the DNA base ratio because it is diluted with inert or structural RNA. Beyond

the

Template

Many scientists are tempted to look beyond protein synthesis to the darkest area of the over-all picture. How does DNA transmit its information on protein structure to template RNA? Another intriguing question: How is information on the RNA template conveyed to sRNA so that it knows a particular amino acid? Dr. Hoagland, Dr. Zamecnik, and Dr. Stephenson offer some speculation on both these points. There is evidence that template RNA is synthesized in cell nuclei. One possibility is that DNA acts as a template to make a double-stranded RNA. Alternately, DNA may synthesize a single strand of RNA, which can replicate a complement of itself. MIT's Dr. Rich has shown that a synthetic DNA chain can form complexes with one or two strands of synthetic RNA, thus showing that a DNA-RNA complex is possible in the nucleus. Dramatic evidence that DNA does indeed play an important role in RNA synthesis comes from Dr. James Bonner and co-workers at California Institute of Technology (C&EN, April 24, page 60). Using DNA-containing nucleoprotein from pea embryos, the Caltech group has synthesized RNA from its four precursors—the triphosphates of adenosine, guanosine, cytidine, and uridine. Other aspects of this finding underline its importance: • Both DNA and protein of the nucleoprotein (as well as other, unknown components) are necessary in this cell-free system. • The newly made RNA is inti-

mately associated with the DNA of the nucleoprotein. It even sticks with it through a procedure that strips away the protein. • When complexed with DNA, the RNA is not attacked by ribonuclease. But this enzyme will degrade the RNA when it is released from the complex. These results support the idea that DNA may transfer its genetic information to RNA by directing the synthesis of RNA in the nucleus. If RNA exists as two complementary strands, Dr. Hoagland and his coworkers theorize, one of them may be broken down by an enzyme into soluble subunits. These then go forth to react with amino acids, after having a terminal triplet C-C-A attached by enzymes. The feasibility of this last part has already been demonstrated by Dr. Zamecnik's group, by Dr. Van R. Potter and his associates, and by Dr. E. S. Cannelakis. The sum of their work indicates that CTP (cytosine triphosphate) and ATP are precursors of the terminal C-C-A of sRNA molecules. It is possible to remove this terminal group from sRNA. The resulting sRNA can't accept amino acids unless CTP and ATP are present. Some enzymes are probably involved in the reaction that puts them back onto the sRNA. Since the fragments leading to sRNA originated in one of the complementary RNA strands, according to the theory, they must have the configuration needed to recognize their places on template RNA. Thus, the theory provides for genetic continuity from the DNA to the microsomes to the events in the soluble phase. Even though today's biochemists work with systems that are vastly better than those of a decade ago, the present systems are still too poorly defined to give the specific information that is needed, many feel. What chemists would like to have is a system that can be fractionated into the individual enzymes, nucleotides, and other components essential to the scheme. They are making steady progress in this area. The closer they get, the better able they will be to answer the remaining questions about protein biosynthesis. The doubtful and unknown parts of the mechanism still outnumber those areas which have yielded concrete, meaningful data. One important problem: Has proContimied on page 89 MAY

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CHEMICAL SCIENCE SERIES Continued from page 87 tein actually been synthesized in any of the cell-free systems commonly used? Many chemists feel that it has. But they point out, too, that rigid proof has been hard to come by. Ex­ periments designed to show this usually use C 14 -labeled amino acids. The radioactivity shows up associated with the microsomes or with protein isolated from them. The difficulty here is that the amino acid may not actually be a part of the protein chain. Also, the protein iso­ lated may not be the specific one that is made by the microsomes in vivo. Amino acids tend to attach themselves to many types of large molecules. There are many limitations and pit­ falls to this kind of experiment, chem­ ists point out. Research in various laboratories is aimed at answering such major ques­ tions as:

• What is the smallest segment of sRNA necessary for the specific reac­ tion with an amino acid and its ac­ tivating enzyme? • What is the code that allows sRNA to recognize its position on the RNA template? Where is the code located on the sRNA chain? These problems must await the isolation of pure, specific sRNA's. Then chem­ ists will be able to take them apart piece by piece, an essential step in answering both questions. • What are the steps lying between formation of aa-sRNA and the final protein? What enzymes are involved? • H o w does GTP fit into the final step? What is its fate? • What triggers the release of fin­ ished protein from the template? • Are different microsomal RNA's necessary to synthesize different pro­ teins in a given cell, or does a single RNA chain contain several templates, bringing the desired one to the micro­ some's surface when needed? • How does the cell fold the pro­ tein into its specific shape, introduce cross-links, and attach nonprotein molecules? The broad scope of these and other unanswered questions points up one hard fact: Many years of difficult re­ search lie ahead before the mecha­ nism of protein biosynthesis is clear. Scientists who work in the field are the first to stress this point. Right now, the over-all picture of the mech­ anism is mostly theory. Here and there, experimental data brighten small areas. But the combination of fact and theory provides a framework on which to design tomorrow's experi­ ments. The most exciting findings are yet to come.

COMBINED REPRINTS . . . . . . of this article, together with Parts II and III to be published in the next two weeks, will be available at the following prices: One to nine copies—$1.00 each 10 to 4 9 c o p i e s — 1 5 % discount 5 0 to 9 9 c o p i e s — 2 0 % discount 100 or more copies—rates on request Address orders to Reprint Depart­ ment, ACS Applied Publications, 1155 16th St., N.W., Washington 6, D.C.

MAY

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