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Illinois Wesleyan University Digital Commons @ IWU Honors Projects Biology 1964 Nucleic acids and protein synthesis Sheldon Nicol Illinois Wesleyan University Recommended Citation Nicol, Sheldon, "Nucleic acids and protein synthesis" (1964). Honors Projects. Paper 47. http://digitalcommons.iwu.edu/bio_honproj/47 This Article is brought to you for free and open access by The Ames Library, the Andrew W. Mellon Center for Curricular and Faculty Development, the Office of the Provost and the Office of the President. It has been accepted for inclusion in Digital Commons @ IWU by the faculty at Illinois Wesleyan University. For more information, please contact [email protected]. ©Copyright is owned by the author of this document. NUCLEIC ACIDS AND PROTEIN SYNTHESIS b� Sheldon Nicol Senior Honors growth of among biochemists a result of, a:ad has contributed to,. our adva:acing o f the chemistry a:ad Over the past two intimate J.�"'�iJ..!.\LJ.J of the , evidence has accumulated Q�.V;�� an between cellular nucleic acid and protein A large number of experiments in shown systems that ribonuclease disrupts the cell's protein synthetic that ribonucleic acid (RNA) ca:a frequently restore it. Studies on bac terial tra:asformation ( Hotchkiss, 1957) and the discovery of the autonomous infectivity 1956) tobacco mosaic virus RNA ( Gierer the neces- that nucleic acids to direct on body of demonstrates conclusively of acids particles ( ribosomes) . was received much to synthesis was another, and a -2- he He a and aware o f , and he was But he of of its function, because the no at the time, even to Mendel. the nucleus in heredity was It was well over half a century later, long after the death of both men, before their work could be fused. The fusion required much more nucleic acid and protein chemda�ry than existed Miescherts time and a vas� of new biologyt including the later unearthing of Mendel's work" During the last thirV years of the nineteenth century, work in biology, led by Weism�, suggested the continuity of the germ plasm heredity" and implied that the nucleus of the cell plays a central Attention soon equal chromosomes.. on ,",UWU.L",lJlJ.=A Since sperm and egg uY.'v.l.<:,.I. chromosomes and of and are concerned with and the redisc overy £h",�'I!!rI�h more .. from a .......,""-,,,...... far more that became onY.U.VIlJ;LV The than the genes as "' ...1--,....,.,''', were shown to contain the was of The now that DNA came in the a given organ- cells was measured a.nd found, of chromosomes" be constant per DNA as the essential material of the genes. This pointed to Numerous experiments could be transmitted from one pneumococci showed strain of bacteria to another by transferring, to cells of the latter, DNA extracted from the former. These experiments proved conclusively that DNA is the carrier of genetic information. These developments made a great impression on geneticists, for they went a long way toward answering questions concerning the nature of the genee The impact on biochemists was far greater; biochemists couad now study the molecular baSis of gene action, tracing the effect of DNA in discrete observable events in the synthesis of molecules composing the cell. In the years, it has become possible to molecular level, some of the fundamental questions of genetics and came in 1953, when The most scientists, J. D. Watson and F. H. Go Crick, after for lography, devised a new molecular on a double helix model with x-ray crystalDNA molecule, and Crick, 1953) @ hypothesis of the tetranucleotide For According to this n�7·"",-!cn These results from are ester aJZ,Q'r�"'f:l'I'I."t.e,$l of four each of which was of seemed to ""'Y.I"''''''''y from the combination of the amounts of the in the (2) to of early determinations of the molecular weight of the acid gave figures which seemed to indicate a molecule of about tetranucleotide (3) size, and titration of the acid indicated four or five free acid hydrogens in the phosphate radicals. ture Th�s was best explained by a struc� in which one mononucleotide is linked to another through a phosphate ester linkage from the phosphate of one nucleotide to a hydroxyl group It had even seemed possible on the basis of of the sugar of another. their relative rates of hydrolysis to decide Which nucleotides were the outer ones and which were the oentral ones. In accordanoe with the avail able etidence, various structures were proposed for the tetranucleotide, including open chain compounds and giant rings. The Watson-Crick model for the moleoular structure of DNA is based on the construction of moleoular models arranged to conform to the cal and chemical characteristics of the DNA molecule. .Inthe�.�prop6sed structure, two molecular strands are coiled around a common axis to form a fairly rigid helix.. Each chain is a long polynucleotide, resulting from the linkage of many individual nucleotide units. The are formed by the combination of molecules of desoxyribose sugar with phosphoric acid molecules and nitrogenous bases. There are two bases .. each " DNA:. on the six-membered a and oan thus be are atoms.. and in compounds with two nitrogen atoms bases are ring. or one of two types: The nitrogenous base Two , because it possesses an� DNA: group, .....I.<,.u.<:'u. . can be from e In the T2, and the place of wbeat germ and of tbe cytosine replaced by methyl- Glucose bas been reported as a constituent of the DNA of cer cytosine. tain bacteriopbages (Sinsbeimer, 1954) . Tbe purine or pyrimidine base is attacbed to tbe sugar at tbe one-carbon and the phospbate to the three carbon. The nucleotides are linked togetber by phosphate ester groups in sucb a way that the three-carbon of one sugar is linked to the five carbon of the next. The unique feature of tbe Watson-Crick structure is the arrangement of the bases. The two chains are joined together by hydrogen bonds w�ich join the opposing nucleotide bases. The phosphates and sugar groups are on the outside of tbe helix; the bases are on the inside and directed perpendicularly to the fiber axis. Bonding occurs only between adenine and t�mine and between cytosine (or a substituted cytosine) and 51A.::'U.I.U'='" Adenine-tbymine bonds contain two �drogen bonds, while cytosine-guanine bonds contain three hydrogen bonds. Hydrolysis of DNA preparations yields the desoxynucleotides of adenine, guanine, cytoB�ne, and thymine. There are differences in the base ratios of DNA preparations from,different species; that the molar amounts of adenine, guanine, cytosine, and thymine are not 1:1: 1: 1 of DNA , but there are definite analytical regularities" as required by tbe old tetranucleotide bypothesis to cytosine seems to thymine and one. to equal vary of nucleotide The and are Tbe for each ratio, which • from the so- This ratio of and to 1 The the sea o and l m an no known restriction on the sequence of bases in one chain There but it is obYcious that the chains are complementary; that is, once the sequence of bases in one chain is known, the necessity for specific pair ing determines the sequence of bases in the second chain. tide rotated sive base pairs are molecule 20 36 degrees with respect to its neighbor and succes 3.3 Angstroms apart (DeRobertis, very long (about ��gstroms)in diameter ) , (Strauss, Each nucleo 30,000 nucleotides), 1958) . The DNA quite narrOw (about and has a molecular weight of several million 1960) . The experimental the double helical ..."'" .u.,::;.u. other than lllV'�O"" evi- two evidence. dence and the The chemical evidence shows that the molar ratio of purine and pyrimidine bases is very c:1ose for sources of DNA. There is now much physical evidence to Titration curves support a form that the bases bonds and that these are bonds within the measurements show viscosity, and DNA in at DNA, so that the does not come one there are oppo- and combines The phosphate-sugar backbone repeats regularly, both chemically and structurally. This repitition necessarity implies that the phosphate-sugar groups are related by symmetry, in this case by a screw axis, and i t this which makes the backbone a simple helix. Again, the two separate phosphate-sugar backbones are related to each other by symmetry, in this case by two-fold rotation axes perpendicular to the fiber axis.. The arrangement of the bases, however, does not repeat, a.nd only shows pseudosymmetry; that is, the region occupied by a pair of bases is fixed, and successive regions are related to each other by sym metry, but there is no restriction on which pair of bases occurs at any point t as long as one of the allowed pairs is used e It remains to be seen whether there are structural reasons for the particular base pairings. It sho�ld be noted that while x-ray diffraction shows that a sub stantial portion of the DNA must be in the double helix form it is an extremely poor method for deciding how much of the DNA is in this con figuration. The titration curves and the analytical data suggest that the great majority of bases are paired.. However, it seems certain that the molecule is folded in its biological condition, and that • be occassional regions where';.the configuration is somewhat of DNA with the crystalline desoxyribonuclease results dialyzable fragments, but a non-dialyzable frac- in a , whose '"'VJ... "''-''''' .... ginal DNA and the dialyzate. from the ori This indicates a complex and asymmetrical of the DNA of DNA form fibers with intense and viscosity" 1'1""''''''': microscopy shows that DNA and fibrous indicating the asymmetry. This molecular and the tendency of DNA particles to become oriented under ) stretching is also revealed by the dichroism shown in ultra- violet light. result from the combination of nucleic acids and proteins and, in certain cells, constitute the majorJart of the solid material. Nucleic acids combine with the proteins by ionic bonds which It is suspectedihat a basic protein are dissociated with relative ease. lies in the narrow groove that spirals around the DNA molecule and that the component basic amino acids of the protein neutralize the phosphoric acid residues of the DNA. Two main types of basic proteins have been one of low molecular weight found associated with chromosomes: mine) molecular weight (histone ) . and one of the DNA structure In addition, there .. occurs an acidic protein of high molecular Evidence suggests that the protamine ( prota- ... •••'" ....AJ.."" ....,.,)" wound helically around the smaller of the two grooves between the backbones. Models show that an extended polyarginine chain can be fitted in this basic groups , with the groove without the side-chains going alternately up and d�wn to the negatively charged phos- In phate groups of the DNA backbones.. nucleoprotamine there appears for every phosphate, yet only two-thirds of the protamine be one are basic. whenever the V'l,",'"_''''' to tively easy with two the the This suggests that the polypeptide amino U..I. ..L\"J.J.J.� occur .. "',u,......... shows that , but rela- a fold with one succession.. on the amino acid sequence residues do occur in between the is folded is and the clear of or folds go or outwards. are that ( nor The main features of the preliminary x-rays part of it - maintains its character- the DNA - or istic structure; and that (2) some larger repeating structure is also present. These results are obtained wi th nuclei, swollen in water and into fibers, with artificial combinations of DNA with lysine-rich histone. One equatorial spacing (of about 60 Angstroms) little on drying; another, around 40 Angstroms, alters with hum� dity (Crick, 1951). These results show that nucleohistone has a structure some sort. In the chromosome, each of the ultimate fibrils consists of two cemplementary DN:l\;malecules intertwined about the basic protein. fibrils are 3-4 mu in diameter and several mici:en�.lon:g:�.c (,J..)�UJ.I.L..L";l:>,�JI;:::tnEase .5-1 micron form the microscopically meter. These dia- Whether or not a single DNA molecule extends the entire length the chromosome matter of debate. The chromonema UHli,l"",,_',,",,, characteristic coiling cycle which may be the result of cyclical the chemical links between DNA and�its basic protein partner. VLL'_'�>Q� The behavior of chromosomes m� be experimentally modified by treatpotassium cyanide" ment with metabolic state, the chromonema has a When at a of chromomeres. chromosome Chromomeres form compact path of the chromonema" The of DNA various means has of does occur. been observed in observation A " often 40 form 1'���,mN� and Butler, 1955). or more, of i:ij'C'I.A..l..L!!l::'Ll. observed DNA a specimen Therefore, there must be considerable varia It has not yet been tion of particle configuration Qf DNA specimens. possible to prepare constants, specimens by fractional centrifugation in suf DNA It appears that ficient quantity to determine their composition. does not exist as fairly straight rods of constant diameter and differ�t length, but rather that some kind of aggregation occurs. RNA , general, structurally similar to nucleotide but is generally single-stranded. the sugar ribose of desoxyribose" DNA. It a long poly- In its backbone it contains The bases linked to the ribose units c an be any of four diff�rent types, all similar to those found in DNA except , which of found DNAts " RNA abun- dant in the cytoplasm, particularly in the ribosomes. DNA can betbopographically located within the cell by means of the Feulgen nucleal reaction. This method consi sts of treating the tissue with Schiffts reagent (basic fuchsin bleached with sulfurous anhydride, The after acid a positive reaction (they combine with occur acid to but it s two recolor the basic negative with RNA" This reaction of DNA of the cell In free bases are groups in the DNA the sometimes second reaction between the mole by means of groups and the decolored fuchsin. When the is , the in the cytopl asm. and the the nucleus, the is nega- tive and chromonemata (especially the chromocenters) are intensely pos itive. In ultra- violet light, nucleic acids show a characteristic absorp 2600 tion peak at bases. Angstroms due to the presence of purine and pyrimidine Ultra-violet micro spectrophotometry permits the localization of the two types of nucleic acid without distinguili1 ing liJetween them, the nucleal reaction of Feulgen shows DNA presence. Some time ago it was thought that the double-stranded DNA structure occurred in all organisms. Recently, however, there have been reports of a virus which has a single-stranded DNA structurel' judged from its suscep tibility to inactivation py�'phosphorus-32 decay (an efficiency of one inactivation per disintegration which is about ten times the efficiency of inactivation of the double-stranded phage DNA) and by the unusual physical properties of the nucleic acid (Sinsheimer, 1959). The finding of a virus with a single-stranded DNA implies that the gene tic information can be obtained in a single strand. DNA located exclusively in cell nuclei. cell nucleus is relatively constant. Although the content of drastically with of the cell, the DNA DNA content The amount of DNA per . and the physiological condition remains constant as would be expected if the gene number. and does not It DNA metabolism. ina of shows that among ""'<Nt""''';;.... in size of the are of content lowest DNA and values are found The values coelenterates. tJles, .and birds suggest DNA content the most that in lung of DNA such as sponges primitive fishes, amphibians, during evolution there has been a decline (Mirsky and Ris, 1951) . The;.zceal experimental)ev:irdence that ;f6rces'ex:clusi1le;;c�msideratii.:olb.., genetic mljl,teI!ial; come.s d}Ilom '.studies:;with .:1fliCIlOO rganisms;, fromLs:'bud:j.es on:.the ..tr:anstkll:rmi;p.gfprinciplesEQf1bacteflia... ; Pne umoc ocoi : o rdinarily . possess:;a smooth polysaccharide after oulture the � vitro, is . polysacoharide This oells not and whioh have a transformation has been shown the capsular to be DNAe quite polysaccharides; transformations of good that the eliminate the transforming that protein Although many are transformed an .. The DNA the of is one at a time.. therefore Only a donor DNA the of one set of genes does for of but into the .. of is Transformation donor, when introduoed the It really characteristics at one time. a complete SUbstitution drug principle is plays a spacifio contained in the not have been achieved. 1957) . DNA unoontaminated by protein transformation process. responsible Transformation , biochemioal oharaoteristios, etc. in which rough appearance. The substance develop;t � he ability The evidenoe oolonies in are abtained which yield produ ced Occassionally irreversible; rough cells never spontaneously transformation restricted to c61at. still to speculation, some basic facts have been demonstrated. Most interpre tations are influenced by the Watson�Crick model, wbdbhgives use to the theory of replication by a template (model ) process by which each strand acts as a mold a synthesized one. reproduction, this a positive print. in This .. can be compared ... ,u.."'".... """' which a negative is made first and Since one of the polynucleotide chains determines the synthesis of the other during interphase, all that is needed to set the template mechanism into action is the s�paration of each strande This hypothesis presupposes that the simple nucleotides fall into phase and that the parent helix of DNA unwinds while the daughter helices are formed <and unwound" Calculations indicate that energy requirements for such an uncoiling �e not too great. Nonetheless, a detailed model for the duplication of the coiled DNA as it occurs �� difficult to make even though there have been several attempts. In nuclei of living, proliferating cells, a violet absorbing component has been found which cells. ultraabsent in non-dividing It is postulated that this component comprises relatively unpoly merized DNA precursors that are lost by fixation.and hydrolysis. This ; it in the metabolism of folic the of Some which is a precursor of coen;" act on the may produce a chromosome A deviation�from normal rfl�r��nc:e with the from. has been few DNA (Kornberg, 1951)3 The synthesis deteoted by the inoorporation of labelled desoxyribo- nuoleotides into DNAuus1ialg a polymerase enzyme preparation obtained from Esoheriohia .2.2.ll- oells. reported. A net syntheslis of 50% or better has been all The enzyme system has the following characteristios: four desoxyribonucleatide triphosphates are required for appreciable synthesis (the diphosphates will not substitute), magnesium required, the system is essentially irreversible (as would be required to explain the metabolic stability of DNA), and about 1% of the maximum reaotion rate is obtained when a single nucleotide is eliminated from the reaction mixture. Polymerized DNA required to serve as a primer for the reaotion, upon which complementary chains are to serve as the constructed. Overall proportions of the bases in the product DNA the base composition of the primer and the sequence of the the DNA. the same as synthesized DNA _�':;w..&l"''''''''' Incorporation ratios of their concentrations in the reaction ""'"""7'." is a In cells of the the by as is the case for the an The in LL�!U���- RNA (Oohoa and is increased by strand of DNA serves as a model. to the llJ.'M;!.l.�jJ.· the chromosomes are still in their extended the form and are not that the lll .l.\... U..... v method of the There not a continuous activity of the going since this cell set sort of be higher organisms. the of DNA requires the activation of the Kornberg enzyme but there is no knowleq;ge of what sort of signal DNA required. itself cannot be the template material for protein synthesis. The major site of protein synthesis in most organisms of the cell, a region devoid of DNA protein synthesis from DNA. It the small possible to dissociate synthesis; that is, inhibition of the synthesis of DNA by ultra-violet light, x-ray irradiation, mitomycin, or thymine starvation does not inhibit protein synthesis (Spiegelman,",jlf)9��,;,\;;The high concentration of the cytoplasm suggests that this substance RNA has a major role in protein synthesise RNA in in which enzymes are being pro- rapidly duced. cells and in in Cells involved activities that do not include protein synthe sis contain very small quantities of RNA, inactivates RNA. The DNA RNA, out to the cytoplasm (to the w hich The template RNA mole- to direct are produced from e and protein synthesis the genes controls the production of a specific RNA template or in Ribonuclease, an inhibits protein synthesiSe The relationship between the genes, now concentration is always high DNA molecules by specific base The amino are to the template RNA molecules by low molecular appears to occur the in the There have been just as molecular transfer and a ) seems to occur in a system other than the ribosomes .... .1,..... ",..,.... and Ochoa, 1958) but in higher organisms the synthe- of protein seems to take place m ainly in the ribosomes. Ribosomes appear as uniform, round, electron-dense particles having an average diameter of 100 to 200 the total cellular RNA (Hoagland, Angstroms. (iThe.l.rio<fS0meS}Contain 80 to 90% of 1969). They exist apparently free in the cytoplasm in some tissues (notably bacteria and rapidly growing mammalian cells) , perhaps sometimes attached to the limiting membrane of the cell (bacteria), usually arenfound in association with lipopro- tein-rich membranous material of the cytoplasm. The ribosomes, in natu- association w ith this membrane-like material comprise what has been variously named the cytoplasmic ground substance, ergastroplasm, or reticulum by electron microscopists. of individual The on the ribosome may be bound to each other particle protein by magnesium ions and base interaction for that RNA is the synthesis is the ability of tobacco mosaic virus RNA to direct the forma tion of a specific protein and of the RNA from certain animal viruses to infect and lead to the production of complete virus"containing protein , RNA 1959). There are many other experiments which show that formation. must For "'A':;w.u�)..."" thymine-requiring bacteria can still form induced enzymes in the absence of factor and 1955) , with the cannot. formation of or RNA sis on RNA can occur the absence of The direct between DNA and RNA has been demonstrated The enzyme RNA polymerase, when by experiments with cell-free systems. placed a cell-free medium containing the four nucleotide units of RNA, Addi fails to induce RNA synthesis even when RNA is added as a primer. tion of a small quantity of)DNA, however, results in rapid synthesis of RNA. Thus it is actually DNA that stimulates RNA synthesis. is appar- ent that the four nitrogenous bases appear in the synthetic RNA in the same proportions as they oocur in the DNA molecule used as a primer, with uracil substituted for thymine. Further evidence of the specificity of the reaction is obtained by using as primers two synthetic DNA polymers, one containing only thi)'1Iiline (poly-T) and one containing alternate units of and thymine (AT oopolymer). the With poly-T as the DNA , RNA contains only thymine's regular partner, adenine. With AT copolymer as the primer, the. RNA consists of a copolymer of uracil and adenine in perfectly alternating sequence. even all four the reaction (Hurwitz and Furth, 1962). by DNA is also seen in living cells. bacteria, RNA an host This specific are The control over RNA synthesis When bacterial viruses infect formed that resembles the virus DNA and not that of base composition (Volkin, Astrachan, and Countryman, 1958). One can conjecture the helix of molecule in the C!DUrSe of the reaction so that ribonucleotides (in triand off the the aid of the enzyme RNA one strand of the DNA. of the the ribonucleotides to then from the DNA groups up into an RNA polymer. The RNA and thelDNA reverts to its previous double-stranded form. The of a messenger RNA sent out from the genes to the ribosomes been confirmed by experiments with T4 virus. Colon bacilli were grown on SUbstances containing heavy isotopes of nitrogen and carbon. The cells were then infected with T4 virus and simultaneously transferred to a medium containing ordinary nitrogen and carbon. :Imroediately after infec tionthe(cells were exposed briefly to radioactive phosphorus to label the messenger RNA produced by the virus DNA. two ribosome fractions After centrifugation, the examined for radioactive messenger RNA. It was found that only the heavy ribosomes (those present before infection) were radioactive (Brenner, Jacob, .and Meselson, 1961). new ribosomes were synthesized after infection. Therefore, no: Ribosomes, then, are responsible for synthesizing protein. Not until the machine has been supplied with the proper instructions, supplied by messenger RNA, can a be • Developments in understanding the cell-free system illuminate another, and more direct, participation of RNA in protein synthesis.. the soluble, nonparticulate fraction (pH 5 fraction), found for the overall incorporation of amino acids acids by formation of enzyme-bound amino of reaction mnner. The compounds from that and amino same ribosome the carboxyl activation of amino that (adenosine It is known amino acids activating enzymes not a the amino thus but to acid of RNA molecules. , low mole- These molecules, once charged with amino acid, are able to transfer the amino acid to the protein of the ribo nucleoprotein parti�les. Ultracentrifugal and electr�etic analyses of transfer RNA (sRNA) preparations indicated that they are heterogeneous. It is apparent that sRNA must consist of a number of distinct acids ii&;re attached to sRNA additively and terminally" A characteristic of transfer RNA's is their content of a relatively high proportion of unusual bases, particulary pseudo-uridine, of which one or two molecules per chain are present 1960). dilic, (Dunn, Smith, and Spahr, The amino acid-carrying terminal is uniformly cytidilic, cyti adenylic acid in all 20 amino acid"specific sRNA's, while the phosphate terminal of the chain appears to be uniformly guanylic acid" The of 30,000, corresponding to molecular 6 10 20,000- little 65-85 nucleotides comparison, the high template RNA molecules have a molecular U960). Protein synthesis is often measured by the incorporation of radioactive amino a into the (ATP and guanosine of 5 lLUL/U«J.J. seems to be There is in and medium of the many other ""T,,,+,,,m,,, have been the best results. that the The the of amino are ( of into the are incorporated have ��'JQ"m�'� protein the of reticulocytes. a ratio characteristic of the com- of hemoglobin, the protein produced by these cells are not incorporated protein. system. The amino � vivo, and the ratio of the total amino acids in the ribosome This indicates that a specific protein is being formed by the It is important to show that amino acid incorporation really represents protein synthesis sinc� in some systems, it was thought that the process of amino acid incorporation could be dissociated from true protein synthesis although the processes were related. The first process in:pprotein synthesis consists of the activation of amino acids. The process of peptide formation from free amino acids is not spontaneous and the amino acids must therefore be converted to compounds with a chains chemical potential. The lengthening of does not necessarily require large amounts of energy and can occur by transpeptidation. This process depends on a supply of peptides and can be catalyzed by the proteolytic enzymes. known It is not amino acids has contain both amino and carbolKiyl groups and since both types of groups can be activate�it was not obvious how the activation would The evidence now is that activation occurs by reaction of the cargroups of the amino of as of the group which • This was suspected because of the beha- that form simple peptides and could for protein synthesiSe Such systemslare the the acetylation of sulfonilamide, and t he In be of all these A or by of At synthesis the ATP to form an adenylic acid deriva- amino acids tive of the acids the combination of which the c arboxyl group of the amino acid of the\'A:TP G is combined with the In the presence of enzyme and amino acid, phosphorus-32 labelled ATP will e�change with non-labelled pyrophosphate� this reaction that all 20 involved in protein synthesis One evidence that the demonstration of the common amdno acids can be activated ( Lipman, 1958); another is the behavior ofcertain analogues of trypophano Certain ana logues of the amino acids are actually incorporated into protein. (1951) and Lipman Sharon demonstrated that those tryptophan analogues which were incorporated into protein were activated by the tryptophan-activatenzyme. Those vated but which were This correlation indicates inhibited that the activation reaction acti- incorporated were a part of the protein synthesis mechanism. Activation occurs in the soluble or supernatant fraction of the cell. is, each amino enzymes are vated by its own single specific enzyme. vating enzymes Several relatively pure acti been isolated and the work continues. amino acids do not compete for enzymes that activation is entirely The fact all sites indicates be located. There is of RNA. This affect the based on the fact that amino acids by crude or enzymes the more RNA are fully not the absence of RNA. The bou..ll d to a very small acf�81)"[;O:I'" for the amino amino of an the Thus the activating enzyme and bound can be thought of as acting effectively as a single The of amino acid to sRNA is mediated directly by the activating enzyme without the involvement of other enzymes. protein synthesis, the amino acid becomes In the second phase attached ester bond to the terminal adenosine of the an corresponding sRNA 1960). Glassman, and Schweet, This RNA have built into its nucleotide sequence a specificity for the particular amino acid to which it becomes attached on the particular activating enzyme. It is important that the reactivity of an sRNA with its eor responding amino acid varies with enzymes from different species. transfer RNA for a particular amino affinity to the " in ester by for all therefore mot enzyme, now well known that The amino acid is joined on the sRNA to one of the adjacent free hydroxyl groups of a of The third step in the synthesis of proteins is the transfer of the amino acid f�om the sRNA to the template RNA of the ribosome" that been demonstrated The RNA reacts fact that sRNA A into seen for sRNA and to deserves a cyclic, coenzymatic the in the complementary.sequence of combines ��'�\JQ�.ll� RNA for is It has for very broad in code a CWoJ.... the "''''....''' world" The amino RNA, therefore, seems to carry two apparently separate specificities: the activating , and (2) for position on ribosome.. ( for Work with the electron microscope has provided a direct and remarkable view of appears rib0somes in the act of synthesizing the protein hemQglobin" that several ribosome particles are linked together at the time of synthesis, probably by messenger RNA» 1963). Knopf, and Rich, It has been proposed that the ribosome attaches to the messenger RNA at one end and travels across to the other end. In the process, the ribo some synthesizes hemoglobin according to the code it reads off the RNA molecule .. The coding problem can be explicitly stated as problem of how the sequence of the four bases in RNA determines the the protein. acids and one " The problem has two aspects, one general Specifically one would like to know just what sequence of bases codes each amino acid. has 20 of DNA units, of the do w ith the they are arranged in the The more general aspect of the coding molecule, and the way in Which m essage The simplest sort o f code would be one in which a small group of stands one particular 16 this would More the One and at code group " a codon" This gro�p can scarcely be a A group o f such a code .I.J.,=,,'::ou<::'u. which would a bases that codes one amino schemes 20 are called that only certain can consequence leads to a change in that in amino acids. cG,de seem unlikely G Recent evidence makes In the first place, there seems to be no restriction of amino acid sequence in any of the proteins so far examined. has also been shown that typical mutations change only a Although it 1'3 amino acid in the polypeptide chain of a protein" theoretically possible that the genetic code may be partly overlapping, � more likely that adjacent codons do not overlap all. Since the backbone of the DNA molecule is comp�y regular, there nothing to mark off the code into groups. To solve this difficulty, various ingenious solutions have been proposed. It was thought, for example, that the code might be designed in such a way that if the wrong non- of triplets were chosen, the message would always sense and no protein would be produced" message and But it now looks as though the at a fixed starting pmint, probably one end of the gene, read bases at a time. If the started, at the wrong point, the message would fall into wrong sets of three and would If this idea were correct, it would immediately explain why the or deletion of a base most of the gene would make the message the reading of the from point onward would be incorrect. In with were always without them were of a base and one causing Il)ocur on the If addition and a , the gene would work" a, base molecule, the gene are too far apart, the be To understand 64 t the code must again be 20 triplets but only to amino acids to be coded. two or more triplets may stand for each amino acid. e There are Conceivably On the other hand, reasonable to expect that at least one or two triplets may to start or to stop protein other meaning, such as a formation. �o'n��'_ Although such hypothetical triplets may have a meaning of some sort, they have been named nonsense triplets. It is possible that the misreading produced in a region lying between an?i::eLddition and a dele tion might give rise to a nonsense triplet, in which(case the gene might work. Investigations w ith a number of addition-with-deletion com binations in which the intervening distance was relatively short have that these combinations were indeed inactive when they might be to function. Presumably an intervening nomrense triplet :to In confirmation of this hypothesis� it has been possib�e to modi�y blame. that turn one base into another, thereby such nonsense triplets by restoring the gene's activity. At the same time it has been possible of nonsense mes- It is read from a fixed point, most likely one end ,iof the So far, all the evidence has fitted very well into the the read in groups idea one end. starting The same results should have been._,�u�'�J'QU' however, if the message. had been read in groups of or indeed in groups of any size. addition mutations were this, either of the gene. " But when all three are The Same fairly close together in one gene. function will or in have been To test the s� gene, by three base dele- tio ns ( Cr ick , 1 9 62). o nd of wr o ng r e ading. w il l br ion will t co d e , B u t if the c o d e is a tr , the me s s age b a ck in w il l b e r e ad cor r e ctly . ion w ill ion is obvious. The a third a d d itio n and fr o m the n o n to the e n d it A l tho ugh the m o s t l ik e l y e x p l anation is that the me s s a ge is r e ad thr e e b as e s at a time , this is n o t comp l e te l y cer tain . T he r e a d in g could b e in multip l e s o f thr e e . T he f ie l d o f ce ll ul ar pr o te in s is and the ins e p ar ab l e f ie l d o f nucle ic a cid chem is tr y w il l continue 0 and n e w inf or ma tion will cer tain l y b e d is co v e r r ap id p ace . A t the pr e s e nt tim e , the m o s t inte r e s t. b e w id e ly s tu d ie d �t a n incr e a singly the co d in g p ro b l e m is pr o b ab l y Dis co v e r y o f the c o d e s f or s p e cific amin o acid s w il l a l l ow e x p e r im e ntatio n with a l te r nation, by chemical means , o f g e n e tica l ly inhe r ite d char a cte r is tics . A pr o b l e m in am.:L"LO a cid co d e s is the d if f icul ty in nucl e ic a cid de mapp d a tio n . a- T his d if ficu lty could p o s s ib l y b e a l l e v ia te d b y s e par ation of d if f e r e nt p ur e DNA s . C hr omat me tho d s o ccupy a r ather ion u niqu e p o s ition among r e ce nt d e v e l opme nts in the f ie ld o f s e T he ter m chr omatogr ap hy now cov e r s se v e r a l m e thod s . s: cie n t lab or atory m o l e cu lar s ie v e , gas , ads or p tion, tio n . in e ions with a s o lu- ctr I a mate r ial must b es. I on ionic tic ion time at ther e f or e , p is b a s e d on the of a s ol id mater ia l To be us e fu l a s an ion co l umns . I u s e d ion chr omat o f ioncexc comm o n io n and e l e ctr o chr omat F or my e xp er im e nts with DNA , T he pr in c par tition , e f f i- cr o s s -link e d or and inor r s ins ar e e la s tic Is that ab s or b er other s o l v ents and in in s erior o f a res in c on c entrated elec tr 11 c o s r f The p ro perties o f the p o lymer e s ol u tio n . n etw o rk , inc l u d in g its l ow d ielec tric c ons tant, a f f ec t the ion b ehavior� and , in a d d itio n , the po l ymer a c ts to a c er tain extent as a s ol v en t o r a d s o rb en t f o r certain c om p o u nd s . I on exc hange c ol umns a re u s ually o p erated by elution d ev el opmente I n elu tion c hroma to grap hy , the s u b s tanc e to b e s ep arated is f irs t s o rb ed on a narrow band o f res in at the top o f the c olumn . I ons o f this s u b s tanc e a re then elu ted , o r c a rried d own the c o l umn , b y a s ec o nd s p ec ies of ions whic h c ompetes f o r b onds o n the res in . ep aratio n is a c c omp l is hed b y v a ry ing the conc entratio n o f the c ompeting ions . F ra c tio n s are c o l l ec ted at the b ottom o f the c o l umn . T he a p p l ic ation o f ion to elu tion c hromat iv e p ro b l ems in the nuc l eic a c id f ield ic a 1 a nd pr d irec tly f o l l o w ed the inv ention o f the t tio n to in o r ! c at Khym , and its and C o hn , 1 9 4 7 ) and w a s a c tually the w a y i n w hic h true el ution c hr was introd u c ed into b io c hemis try . exc ha nge T he c omp l exes b etween the p o l yv a l en t ino rganic c ations a nd the p or a c id s that p erf o rmed s o s p ec taCUl a rl y o n the f irs t tes t o f ion exc c hromato gra p hy may be c on s id ered a s d egree and k ind o f range by teric , inc o f c ha r contro l to them w ithout the nec es s d irec ion s . o v er a w ide ;;:lin c e the nuc l eic a c id s a re a l so it was a p p a rent that the c inasmuch as the may be v aried c an t a d j us tmen t . c o ntro l could eric, It als apparent tha t s ep arations of ith anion o r c i on w ere f eas i bl e. o n uc leic ac id f ie o ( Cohn , 1 9 4 9 ) lie t t ouc h ed of f a s eries of d i s c overies of n ew n uc leo t id es , b ot h t hos e d eri ved f rom nuc l ei c a c i d s t ruc t ure, a s wel l a s I t als o mad e a va ilab l e a c h em i c a l t h os e t ha t exi s t free in na t ure. t ec hn i que of b road us e f ul n es s in a w i d e varie of analy t i c a l and i on p rob l ems . I n my experiment s , I s by ammon i um hyd roxid e elut i on, t o ef f ec t a s ep a ra t i on of DNA, on an i on exc h ange c ol umn . Th e c ol umn was prep a red b y p ouring a s lurry of Dowex- 5 0 , a c ommerc ial c a t i on - t y pe exc ha n ge res in, i n t o a b uret p l ug ged a t t h e b ot t om b y a wad of glas s wool . T h e c ol umn was w a s h ed w i t h d i s t i l l ed w a t er A small q uant i t y of DNA, t o as s ure a neut ra l p H . admini s t ered t o the t op of the c ol umn . inc reas in s ol ut ion , was T o a c c omp l i s h elut i on , c on c ent ra t i on s of ammon i um hyd rox i d e were ad d ed t o t h e t op o f t h e c ol umn . F rac t i ons w ere c ol lec t ed a t t h e b ot tom of t h e c ol umn ( el ut i on rat e = 2 ml . /m in . ) in w e s ampl e b ot t l es . A f t er t h e c ont a ined s ol ut i on was evaporat ed i n an oven, b ot t l e was eac h s amp l e A f t er and the mas s o f t he DNA was c eac h s amp l e was el ut ed , t he c ol umn was regenera t ed b y p a s s i n g t h rough d il ut e hyd roc h l oric a c id and t h en ri ns ing w i t h d i s t i l l ed wat er unt i l a n eut ra l was ob tai ned . F or t h e f i rs t c ol umn, ap p roxima t ely 0 . 3 g. of c ommerc p rep ared DNA was d i s s olved b y h ea t and s orb ed on t h e c ol umn ( of el ut i on c on c ent ra t i ons f ra c t ion 5, 0 . 10 f ra c t ions in a 0 . 05 M . . ; f ra cti on 6, 0 . 20 1 . 00 .; f ra c t ion s Th e mm . ) . used: s ol ut i on of s ch edule f rac t i ons 1 -4 , o. M.; ! f ra c t ions o. 2.00 M . ; f rac i on . ; t he f o I l t re s id u e s w e re o b t a i ne d : f ra c t i o n 1 2 3 4 5 6 7 8 \9 10 11 12 13 14 15 16 F o r t he ne xt c o l umn, 0 .1 g. 0 .0 2 Ivl. - o. g. - o. - 01 0 . 00 3 8 0 .0037 0 .0 0 3 2 0 . 0 0 27 0 . 0037 0 .0 06 3 0 .0 0 3 9 0 . 0017 0 .0 0 3 1 0 .0 24 5 0 . 00 4 7 0 . 0037 0 .0 0 1 2 0 .0 0 23 o f DNA w a s d i s so lv e d b y he a t NH4 0H a nd s o rb e d o n t he c o lumn ( 3 5 mm.). re s o lve t he l a rge i ni t ia l DNA re s id u e c o l umn i nt o pos s ib l e c omp o ne nt was u s e d : f ra c t io ns , 0 .0 2 , 1'1. f ra c t i o n 7 , 0 . 10 H . ; f ra c t i o n 8 , f ra c t i o ns 1 2-1 4 , 1 .0 0 1 4 .8 0 M . S e v ent e e n m I . . ; I n an att in to o b t a ine d in t he f i rs t t he f o l l o wi ng e lu t i o n s c he d u l e NH 4 0H; f ra c t i o ns 4-6 , 0 . 0 5 H . ; 0 .20 H. ; f ra c t i o ns 1 5 -1 6 , f ra c t i o ns , 0 . 50 Iv[. ; 2.0 0 M . ; f ra c t i o ns 17 -18 , f ra c t i o ns w e re c ol l e c t e d a nd t he f o I l re s idue s w e re o b t a i ne d : f ra c t i o n 1 - 0 .0 6 4 1 g . 2 - 0 . 0035 3 - 0 . 0015 4 - o. 5 - 0 . 0010 6 - 0 .0010 7 - 0 . 00 06 8 9 10 11 12 . - 0 . 00 8 5 - o. - o. a c ti o n - 0 . 00 1 4 F or t h e t h ir d c o l umn , 0 . 1 g. o f DNA was d i s o l v e d in h o t wat e r and s or b e d o n t h e c o l umn ( 4 0 mm . ) . t o r e s o l v e t h e ini t ia l p .; , int o p o s s ib l e c omp o ne nt frac t i o n s 1 - 6 , 0 . 0 2 f o l l ow ing e l u t i o n s c he du l e was u s e d : f r a c t i ons 7 - 9 , 0 . 05 I n s t il l a n o t h e r a t t e mp t t he 1'1 . NH40H ; fr a c t i ons 1 0 - 1 2 , 0 . 50 M . ; fr a c t i o ns 1 3 - 16 , 1 . 00 M . ; fr a c t i ons 1 7 - 18 , 2 . 0 0 M . ; fr a c t i o ns 1 9 - 22 , 7 . 4 0 M . Fr a c t i o ns o f 6 mI . wer e c o l l e c t e d and t h e f o l l ow ing r e s id u e s w e r e o b t a ine d : fr a c t i o n 1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 17 19 20 21 22 - I n a l l t hr is is nt 1 . 00 c 0 . 0 0 1 9 g. 0 . 00 28 0 . 0 4 24 0 . 0 0 22 0 . 0009 o. o. 0 . 000 6 0 . 0010 0 . 0007 0 . 0005 0 .0 0 0 9 0 . 0 0 10 0 . 00 16 0 . 0015 0 . 00 16 0 . 0 0 23 0 . 0019 o. 0 . 0012 o. 0 . 0000 t w o app ar e n t c one ar e n o t e d . i o n s and l ar ge smaller p e ak no o ther ic ab l e or c e s e nt in r s id u e small frac - t i ons . o D NA s o f d i f fe r e n t c h emic al sc dule , c o uld ass d own int o s ma l l e r e par a t i o n o f D NAs by c hr oma t o gr ap hy h a s b e e n pr e v i ou s ly a c c o mp l i s h e d b y v ar i o u s me a ns b u t , b e c au s e b u f fe r s o l u t i o ns ar e u su a l l y u s e d , t h e D NA fr a c t i o ns ar e mix e d w i t h s al t r e s id u e s wh i c h make b as e s e qu e nc e d e t e r m i na t i o n b e d e gr ad a t i o n d i f f i c u l t . 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