In Press Cold Spring Harbor Synposium 1963 inactive as terrplates -5x p~o1~4.n sy~tliesio, These ~csKt.ts suggtst thee ?3M with a high propor"iion of helical s t~uctcse may have little `ieqlata activity for protein synthesis, Recent expcF%nE!llt6 have Ght317n that posy U-poly A helices do nolr bind to ribosones, and foi: this reason may be unable to direct protein aynthes3.s (Cukier and tTircnberg, unpu'blfshed F@SUllS). It also is possible that small, localiecd areas of ordered structure may serve aa periods in protein- synthesis. St is diffl-cult to compare directly the messenger effic3xnctcs of different polynuclcotide preparation s because the efficiency Is modified by molecu3.ar size and secondary gtmckuse, However, Ff the average chai_n length and secondary structure of differcx& IXJIA preparations arc assmed to be qqmdnw+ely wpl, the d%al ef Table 1 mgg%st t!m nucl%otide 0cPzgteat my mot bIfLueslce greatly the twerrell template efficdawy sf aWAo Polg 0, ply UC, poly ACG end poly UACG cmtai@ 1, 8, Z?, and 6d triplets reapestiwly, md the preparattons of these polymclsotides &own in Table X have bean fourrd to dire&t 1, 4, 9, md 10 artho aaids, respectively. into protein, The essential point is that agpr0Jtdmetely the fiarm total quantity of amlao paids we tire&ted Auto prateia by each pofymteleotdde, Although them data must be tnterp~eted with cme &ecawe the same fa#stor my not l&nit tlos dneorpcwetbon me of elaclh aho mid, they suggest that the polynucleatide preparatfans nmy hawe appro#imtalgr ergmx tenspme efficifsei638 and thee most aucleotide sequeaet3s my be aI&3 to aode for smino uoidso AlthotaQh ROIIS~PS~ segue~aes aaay exist, thus faq ame hare been demansttated definitively, Colyawleotidos contoinilsg all base eomMnrtfons mw &we bmn wed TV dfrcwt protein synthesis its&u extra~tse A qualitative suamrerpy af these data is presented fa T%ble 2, Only those polpslutleotid8s ContrtiPfng the minimum bases aeawsary to diseet an %&IO scid int@ protein are S~OWBO For example, phenylalaaine is directed into protefa by poly U and other U eon= totniag polymers; however, since other bases are not req&ed, phenyls1aaiae is listed only under poly Us Yoly IJo poly A, and poly C dfrfm pfmylalanioe9 lysim md prolioe, respaetbfely, in%0 protein0 Yolylysine syntheeimi in J& scnlb;b exbraota under the direction of poly A ba$ been found to contain $45 l-ysine residues per lohain (Jones, Bwoa, !30berg Heppei and Nireaberg, B&B- publt shed result& No mrsstibngear mtivity has been deimstretsd fer pely G Watthaei, 9t al p 1%2), bqt t&e highly ordured strrrcture of poly G might msk taplate aetfvityo Hewever, a polyrrucleatdde composed olllly of hypoxmtbine -7- both CCC and CCU would code for proliae and both UUi and WC would code for pheaylafantae. fn Fig. 5 are shown the poly UC-directed eeriue and leucine iucor- poration data. Both seriue and leucine appear to be coded efther by the doublet UC, or by the two triplets UUC and UCC. CocUng of oerine or of lettche by one, rather than 2 tr%pXets is not ;indicatod. St $8 important to uote that if serine is coded by triplets, one triplet would have to contain 2 U residues and the otbar 2 C reeFdues Triplet wozds for leuciue also would contaLn either 2 U or 2 C residtres. These exparimente stxoz@y suggest that bLst2diue, aaparagine, gluttine and lpsiae are coded by triplet words and that the RNA code cannat be cow posed onls of doublets. Threotine, proliue, phenylalau%ne, se'Pine and leucine were found to be coded alther by mult;fple trtplets or by doublets, These data are exnmar$eed in Table 4, A mixed doublet-triplet code cannot be excluded on the basis of the avaflable data; however, a uniform code cou- tainfng only trPplets would appear more probable, The Current Codeword Dictionary. Assambg fox the present that all &no acide are coded by ttiplete, current approximat:iona of R&A codewords my be susmerized a8 shorn in Table 5, Nucleotide sequence is arbitrary, Fifty of the 64 possible ttllplet3 have been assigned, Almost all amiim acide can be coded by polyuucleotideo contaiuing 2 tiffsrout baeeas Sfnce polynucleotidea containing 3 baees dkect protein synthesis as efficiently as polymers contafntng only 2 bases, it seema probable that mont 3 base words are recognized. Tentative aesignments are given for -8- It seems cleat fhat mst ax&no acids are coded by multiple wordsa in base compoelticm by only 1 rzucleotide, These observations also suggest. that mcleotide sequences In mukkple wosds often may be ideuLi+ca'L, A 2 out of 3 uucleotide pairs my, ;In some caseo, ~~uffkx~ for coding9 QT trfplet coda of tUs type in acme. respects would bear a superficial re- semblance to a doublet: code and would be in accord titb a3.l of the data avsUable, The c&ng data obtained thus far clearly %udicate that mst mrcl.eotUe seq?muc%s ~811 code for amino acids vith great specZf%ciCp, Wsfsblm et: al (1962) have reported that mcult%pfe species of leucine tzansfes RNA recognize differen& codewords in synthetic polynwkotides; ?w-wverS additional data suggest that codeword specificity fn dkrectfng lettcina incorporatfon may be greater with aynkhatic palynucleotj.dee than vith rsabual UNA, It is important to enmhasize the poaoib&lfty that xaadcmly-osde~ed synthetic polynucleotides may test the celf'a potential to recognize codewords, and that the entire potential may not be utilized in viva, except perhaps duriq Thus &WA synthesized by a cell may ml: contain as raany codewords mutatfou. 81 randoanly-orded polpmtcleotides D The chemical synthesFs of oli~odeoxymcfeotldeo by the rilethod of Khorana and hts associates (1961, 1962) and the demonstration of an oPigo- deoxynucleotide-dependent syntheoia of polyribomclaotides, catalyzed by ?WA po&erase (Purth et 81, X9613 Stevens, 3961, Chamberlain aud Berg3 1962, Falsechi et ~1~ 1963), provided an opportunity to study their ability to stimulate cell-free amino acid incorporation, Since poly A serves as a template for palylysfne synthesfs (Gardner et al, 2962), oli.go dT (oltgodeoxytbymidylate) has been used to direct poly A: and subsequent 1) ATP se ------"-p Polly A -i- PP 2x1 addition, natural DNA and poly U have been shown to direct polylg~Ine oynthesis, Poly A was symhesized in RNA polpmarase oligo dT reaction mixtures (stage I) as descrgbed In the Legend accompanying Fig, 6, and then components supporting amino acid incorporation Lnto protein (Stage 3X) as in the legend of Fig,, 7# were added* After further incubation, incorporation of C14-lyaino fnto polylyaina was deternlned by precipitation with a TCA - tungstate solution (Gardner et al, 1962), The data of Fig, 6, dzov that C WIMP incorporation was dependent uporr the addtttan of oligo dTl3..14 (13-14 nuclaotiden per chain) to stage f reaction mixtures, and that &%U@ incorporation was proportional to the MIS 242 .- h"Hi LXS a!0 0 Tim w.l cml HIS ACC ii2 cc&a + ACA Gee + CAC 0 cwc w&m4lJcw cm + ccw tmawcc 7 iii !2+ 00 aE 0 70 r+ az * - -0 fj 800 600 400 200 a m-L- ---- _-e---c -4 I I I I I I I I I I 0 IO 20 30 40 50 60 70 80 90 A spamgine Lysine Flj 1 TIME (MINUTES) ------ Theoretied Ffequency of RNA Codewords Observed Frequency of 30 20 IO 0 30 20 IO AC Or Amino Acid hcorporotion AAC+ACC ,/c---v_ ACC "1 / c-a- -1 ---- `\ // 0' . 0 / I I I I I I I I I I A AC /I' /' F-J,`\ \\ \\ \\ \\ ,/// /' I /' .' w . ,-- -`i I I . I I I I 0 IO 20 30 40 50 60 70 80 PERCENT C IN POLY AC 90 80 t cc /I ; CCC+CCA y'// / / or 1 70 // /`ccc // // P 1' /I ,h/ 60 90 00 90 80 70 60 50 40 30 20 IO t \I I- I I I 1 I I I I I I i\ \\ \' \' \' ---- - \' Theoretical Frequency \ `1 of f?NA Codewords \ ' \ ' Observed Frequency of \ ' \\ `1 Amino Acid lncorporolion \\ `\ AA \ `1, AAA': AAC AAAL, `+ \ \ \ \ \ \ \ \ OL I I . 0 IO 20 30 40 50 60 70 80 90 100 b PERCENT C IN POLY AC 60 ooj \ I I I I I I 90 -$, I I I I \\ 80 - `A ------ - Tbeofeficol Frequency 0 of RNA Codeworcis \ `1 70 - `\ `\ Observed Frequency of Amino Acid lncorporo fion 60- 50- 40- 30- 20- IO- % PERCENT C IN POLY UC 30 20 IO 0 I I I I I I I I I I - - - - - Theorefical Frequency of RNA Codewords Obsef ved Ffequency of Amino Acid In corporation 0 IO 20 30 40 50 60 70 80 90 100 5s A- PERCENT C IN POLY UC 8.0 6.0 plus oiigo dTI3.-14 minus oligo d'&3-14 I A I A I . I - 0 1.0 2.0 3.0 4.0 5.0 0 20 40 60 80 100 I20 mp MOLES IpTl IN OLIGO dT13-14 MINUTES 1000 I I I I I I I I I I oligo dTI3- 14 900 - a. 44.8 m p moles - yyo\ I 800 5 g 600 t {- I.2 mp moles 1 minus oligo dT I I 01 I I I I I I I I I I I 0 10 20 30 40 50 60 70 80 90 100 FILj7 MINUTES CHROMATOGRAPHIC POSITION OF LYSINE PEPTIDES POLY- LONGER LYSINE OLIGOPEPTIDES PENTA- TETRA- TRI- DI- I 2400 - 2200 - 2000 - I800 - 1600 - Y ci 3 d 1400 - 0 F 1200 - ii s s; 1000 - l- z 800 - 600 - 400 200 F L -minus oligo dT 0 I I I I I I I I I I I I I I I 0 I 2 4 6 8 IO I2 14 I6 plus oligo dT ; \ plus Trypsin ! I I I I I ,dus oliao dT I . u r - I . B b I WNE INCHES