Reprinted from the PROCEEDINC~ ox' THE NATIOIAL Ac.mm.4~ OF Screams Vol. 49, No. 3, pp. 392-399. March, 1963. THE EFFECT OF SECONDARY STRUCTURE ON THE TEMPLATE ACTIVITY OF POLYRIBONUCLEOTIDES* BY MAXINE F. SINGER, OLIVER W. JONES, AND ~UARSHALL W. KIRENBERG NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES AND NATIONAL HEART INSTITUTE, BETHESDA, MARYLAND Communicated by Robert J. Huebner, January 16, 1963 Current experiments on the synthesis of polynucleotides and proteins seem to us to emphasize the functional importance of polynucleotide secondary structure. Thus, for example, the highly ordered double-helical structure of DNA1 readily leads to ideas concerning DNA replication.2n 3 Although our notions concerning the secondary structure of the various types of RKA are much less precise, it does appear that RNA molecules are primarily single stranded and contain varying degrees of helical cont,cnL4~ 5 Recently, invcst'igators from two different labora- tories have proposed similar helical structures for transfer-RKA6B 7 and bot#h groups have discussed the functional significance of the suggested configura,tions. It is thcrcfore of interest t#o consider the secondary structure of template RSA. Pre- vious reports from this laboratory', s described the template RNA dependent in- corporation of amino acids into proteins in a stable cell-free system from E. coli. Using t(his system and randomly mixed polyribonucleotides composed of various combinations of t#he four common ribonucleot,ides, nucleotide codewords corrc- sponding to almost all of the protein amino acids have been dctermined.1"-15 Observations made with this syst#em suggested t'hat, t8hc secondary at'ructure of poly- mers influenced templat'e efficiency. For example, the ability of poly U t,o direct polyphellylalalline synt8hesis is lost a-hen poly A-poly C double or triple hclices are formtd.st lo The prescnt8 report describes certain physical properties of a series of poly UG prep- arations as well as the eflicicncy of these polymers in directing amino acid incor- poration in t'he cell-frer system. The data indi&e t,hat, secondary structure in a polyribonuclcotida limits it,s efficiency as a t8emplnte. This finding is discussed and a specific funct,ional role for polynucleotide secondary struct'ure in tlhe coding mechanism is proposed. A preliminary account of some of these data has been published.10 Alatericds awl ~~fetkods.-The proredures for the synthesis of polymers, determination of base ratios in the polymers, and measurement of amino acid incorporation have been dcscribcd pre- vicnlsly.sr I1 The UC; polymers are isolated by- a modified procedure I6 designed to concentrate the longer clmin lengt,h polymer and eliminate some of the shorter chain material. At the end of t,he I"`lyrllcrizatiotI tile react.ion mixture is deproteinized by the method of S~itg.~~ The aqueous solution of ~~olyrncr is made 2 Jr in KC1 by addition of an appropriate amount of solid KCl. Polyrnw is precipitated by the addition of 0.2 volume of cold absolut,e alcohol, and collected t)y centrifugation. The precipit,atc is dissolved in a small amount of water. In several cases the polymers did not, dissolve readily in water unless a small anlount of EDT.4 (final concentration about 5 n&I) was added. Precil)itation with KC1 and alcohol is repeat.ed trso more times. The final precipitate is w,nshcd sucwssivcly n-ith SO';;, !)5~~,, and 100 ( ;, alcohol and finally with ether and dried over parallin. Pol,vmers were subsequently dissolved and dialyzed against distilled wntcr before use. The ~~llc~s~~l~~~rc~lysis of the polymers by polynurleotide phosphorylase was determined by meas- uring tile formnt,ion of P3*-labeled nucleoside diphosphate in the presence of Pi32. The procedure referred t,cJ as Assay A by Singer aJld GllSS'* was used; the pertinent polymer was substituted for the poly A of that method. For the phospllorol~rsis studies, ~~~icrococcz~s ipdeikticus polynuclew tide pl~r~sphorylase, purified approximat~ely 250-fold (Fraction VIIILs) was used. Mr:tsnrertlorlts of the t,cmperat,ure dependence of the spectra of polymers ("melting curves") wore carried out eitlw in a Gary recording, model 14M, or Beckman, model DU, spectropho- tometer. The Gary instrument was equipped with a thermc&atted cell holder and the temperature was measured inside the scaled quartz cuvette by means of a hypodermic needle type thermistor. The l~ecknran instrument was equipped with thermospacers and the temperature was estimated in a water-filled, unsealed. blank cuvette. All solutions were gassed with helium just before filling the cuvettcs and the cuvettes were sealed with a coating of General Electric Company ILTV-GO, silicone ruljber compound.20 The chain lengths of polymers were determined by measuring the ratio of tot,al organic phos- phate to phosphate removable by E. coli alkaline phosphatase (Worthington Biochemical Cor- poration). The procedure outlined by Heppel and co-workers21 wa,s followed. Sedimentation velocity studies were performed at 56,100 rpm and 20oC using the Spinco 394 BIOCHEMISP'R I-: SINGEL! ET 24 L. PRO<`. N. A. S. Model E Ultracentrifuge equipped wit,h UV absorption optics. The solutions contained ap- pro.ximntely 0.03 mg of polymer per ml of 0.05 M cacodylate and were 0.1 M in NaCl. For me2tsurements at neutral pH the cacodylste served as a buffer at pH 7.2; fur alkaline measure- ments the solutions were made 0.01 M in KOH (pH 12). Tracings xere obtained from photo- graphic images of the cell using the Joyce-Loeble Recording Microdensitometer. The sedimenta- tion coefficient was calculated22 from the rate of change of position of the 50% point of the bound- ary. Optical rotation wan measured on a Rudolph Polarimeter using the mercury line at 365 rnp and a 1 decimeter light path. Results.- Dependence of amino acid incorporation on the base ratio in poby UG: Table 1 shows the incorporation of phenylalanine, valine, leucine, and tryptophan into protein, measured with a series of poly UG preparations of increasing G con- tent. The relative amounts of the four amino acids incorporated with any partic- ular polymer are clearly related to the proport,ion of U and G. The data show that efficient incorporation of tryptophan requires a greater proportion of G in the polymer than is necessary for incorporation of valine or leucine. These data, t,here- fore, confirm the earlier assignment of codewords UUG, UUG, and UGG t'o valine, leucine, and tryptophan, respectively.ll' 12, 14 It is evident that the efficiency of a polymer as template RNA (Table 1) for any of the amino acids shown decreases sharply when the U/G ratio becomes less than 1. For example, the incorporation of phenylalanine with the polymer of U/G ratio 6.7/l is about 60-fold greater than that obtained with the polymer of U/G ratio, 0.58/l. Although the decreased ability of the latter polymer to direct' phenylala- nine incorporation is expected, the lack of activity in coding for leucine, valine, and tryptophan is surprising. In an attempt to understand this observation various properties of the polymers were investigated and the result,s of these studies are presented below. Chain length of polymers: Each of the polymers shown in Table 1 had a chain length in excess of 300 nucleotide units but probably not greater t,han 500 unit's. Because of the large amounts of polymer required for the chain length determina- tion, more accurate measurements were not made. Sedimentation characteristics of polymers: The sedimentation coefficients of the polymers described in Table 1 are given in Table 2. At pH 7.2 the Sro of the poly- TABLE 1 STIMULATION OF Cl4 AMISO ACID IKCORPORATION BY POLY UG Nucleoside diphosphato ratio (U/G)t 6.7/l 8/l 3.3/l 5/l 1.6/l 0.68/l 3/l 1/l Control minus polynucleotide Incorporation above Control: Cl" amino acid rrrnoles Phenylalanine 901 1708 1067 15 45 Valine 287 1036 1050 Leucine 267 834 856 Tryptophan 38 210 276 10 60 * Base ratio of polymer determined 8s previously described." 4 .. Retlo of UDP to GDP used in polymer synthesis. Flgures represent incorporation of Cl4 of added polymer. amino acid above the basal incorporation obtained in the absence Basal incorporation is Riven in the last column. Reaction mixtures (0.5 ml) contained 0.1 M Tris buffer, pH 7.8. 0.01 A4 magnesium acetate; 0.05 &f KCI; 6 X 10-a M &mercaptoethanol; 1 X 10-a M ATP; 5 X 10-J M potassium phosphoenolpyruvate; 10 pz crystal- line phosphoenolpyrurate kinase (CslBiochem); 2 X 10-a M of each of 19 L-amino acids lacking the Cl" amino acid; 0.8 X 10-a M C'4 amino acid; 5 ~g of polynucleotide; and preincubsted., dialyzed S-30 E. coli extract.9 Each ass&y was performed in duplicate. per millimole. The specific radioactivities of the amino acids varied 2-6 millicuries All reaction mixtures were incubated at 37' for 90 min. These incorporation data therefore represent total incorporation of Cl'-amino acids into protein rather than rate of incorporation. VOL. 49, 79ti3 BZOC'HEMZSTRl': SZA'GER ET AL. 395 TABLE 2 SEDIMENTATION COEFFICIENTS OF POLY UG PREPARATIONS I'olymrr base ratio .%V* - (U/C) IIH 7.2 pH 12 6.7/l 5.1) 5.2 3.3/l 3 1 2 .3 1.6/l 5.7 3 3 0.58/l 11.0 2.1 * Inspection of the dPnsitom&zr tracings indicated rclstively little 01` no breakdoun of t,lre polymem during sedimentation at ~11 12. Nonardinrenting material arnonntrd to al,- proximately ,5'y0. at pH 7 and 12. aitb all the polymrr.3 except thp last (IT/G. O.bSll I, allrn it was about ZOl.:: at pH 12. Furtlr~rmore, in each case thr sedimentine material uns I~OIX homogeneous at JJH 12 than at pH 7 and this was moot striliinq with the last l~~ly~ller (UjC, o.Ss;l). mcr containing TO per cent G is considerably higher than the $0 values of the others. However, the decrease in Sno not#ed for all polymers at pH 12 is much grcat,er fol this high G containing polymer. I'h.osphorolysis of polymers by polynucleotitk ph.osphor$ase: The susceptibility of the poly UG preparat#ions to phosphorolytic cleavage by Al. lgsodcikticus poly- nucleotidc phosphorylase was studied and t#he results are presented in Table 3. TABLE 3 PHOWIIOROLYSIS OF POLY UG BY POLYNUCLE~TIDE PHOSPHORYIASE Base Ratio Fhospborolysis Rate Phosphoroly-sis Ratet (U/G) (nr*molesilS min.) -. Rate with poly `2 poly u* 6.4 6.7/l ii.6 3 2 3.3/l 25.9 3 7 1.6/l 11.6 1.7 0.58/l 1.1 0.16 * Expel.iment carriwl out at a wparate time. t The numbers in tbu last column reprewnt tbr rate of phosphorolysis of the given polymer relative to tbnt of lloly A. The incubation rnixtllrcs contained agyroximately one rmole of polymer ghos~)bate lwr ml (see section on Ala- terials and Methods for details). The rate of phosphorolysis (e&mated wit#h a measurement at a single t'ime when t,he reaction rate is known to be linear with homopolymers) depends on the relative con- tent of uracil and guanine. In particular, when the polymer has more guanine than uracil the rate falls off very sharply. This effect is not, the result of the de- creasing uracil content since t#he members of an analogous series of poly UC prep- arations were all phosphorolyzed at similar rates. Thus, when the ratio of uracil t#o cytosine was 3/l, 3.3/l, 1.7/l, and 0.6/l, the rates of phosphorolysis relative to poly A were 3.3, 4.0, 3.7, and 2.2, respectively. The slow phosphorolysis of poly UG containing more guanine than uracil appears to result' from t,he high guanine content. Effect of temperature on the dtraviolet absorption spectra of poly UG preparations: ThomasZ3 and Doty and co-workers 3, 5 have discussed the use of hypochromicit,y as an indicator of secondary structure in polynucleotides. We have determined so-called "melting curves" for polymers described in Table 1. Polymers having rat#ios of uracil to guanine greater than 2/ 1 showed essentially no change in ahsorp- tion bet,ween 200 and 300 rnh between room temperature and 85'. With poly UG having a uracil to guanine ratio of 1.6 to 1, we observed a slow increase in tjhc absorption at 250, 260, 270, and 280 rnp upon increasing t'he temperature from 22' to 82'; however, the total increase at 270 rnp was only about 5 per cent (Fig. 1). BIOCHEMISTRY: SINGER ET' AL. PROC. s. A. 5. 30 40 50 60 70 80 90 TEMPERATURE ("C) FIG. l.-Changes in ultraviolet absorption of poly UG as a funct,ion of temperature. The ordinate is the ratio between the absorption at 270 rnp at the indicated temperature and the absorption at 270 rnp at the first temperature measured. -a--, poly UG, U/G ratio equal to 0.5811, in 0.1 M KCl, pH 7.8; -O- poly UG, U/G ratio equal to 0.58/l, in 0.01 M potassium phosphate, pH 7.0; -A--, poly UG, U/G ratio equal to 1.6/l in 0.01 M potassium phosphate, pH 7.0. Gnder the same conditions (0.01 M phosphate buffer) poly UG having a uracil to guanine ratio of 0.,?8 to 1 gave a total increase in absorption at 270 rnp of 12 per cent on raising the temperature from 30' to 85' (Fig. 1). The data are given for 270 rnp since the hypochromicity of G containing structures appears to be optimal at that wavelength.24 Figure 1 shows two curves for poly UC (U/G, 0.58/l); one was obtained in 0.1 M KC& pH 7.8, the other in 0.01 M phosphate buffer, pH 7.0. The curves are similar although the start in the increase in absorption is somewhat delayed in 0.01 M phosphate buffer, pH 7.0. This observation is consistent with the finding25 that the "T,," of d-pGpGpGpG is lower in 0.2 M NaCl t'han in phos- phate buffer alone. The experiment in 0.1 M KC1 was carried out in the Cary in- strument and a temperature dependent shift in the wavelength of maximum ab- sorption from 255.5 rnp to 253 rnp was also observed. This shift took place at about 90'. Optical rotation of poly UG preparations: As an independent measure of poly- nucleotide helical content4 the optical rotations of several polymers shown in Table 1 were measured. Determinations were carried out at room temperature in 0.01 M potassium phosphate buffer, pH 7.0, using the mercury line at 365 rnp. The specific rotations of the polymers having U/G ratios of 6.7/l, 3.3/l, and 0.58/l, were +99, + 74, and +399, respectively. Discussion.-Amino acid incorporation into protein: The relative amounts of phenyldanine, valine, leucine, and tryptophan directed into protein by the poly UG preparations (Table 1) is clearly relat'ed to the base ratio of the polymer and VOL. 49, 3963 BIOCHEMISTRI': SINGER ET AL. 397 these data provide strong support for earlier codeword assignments.11, 12, l4 How- cvcr, several propcrtirs of the poly lJG preparations used indicate that meaningful comparisons bctwecn theoretical frequencies of doublet,s or triplets and relative amino acid incorporations cannot be made. One such factor is the secondary struc- ture of the polymers and is discussed in detail below. Another factor is the pos- sibilit,y t~hat~ the polymers may be nonrandom. Since G is incorporated into poly- mer pwfrrcntially (Table 1) the ratio of UDP to GTIP changes during polymer syn- thesis. Thus, the base ratio determined by analysis could represent the average base rat,io rather than t'hat of any particular polynucleot~ide region or molecule. l'refcrcntial incorporation of G into polymers has been not,cd previously,14, l5 and similar observations have been made by Bretscher and Grunberg-Manago,Z6 using ;I. aqilis polyllucleot*ide phosphorylase. The mechanism of this conccnt'ra- tion phc~nomcnon is currently under invest#igat,ion.?' A'J'oct of stwndar,y structwe on th template. activity of meswnger RNA: Several lines of evidcncc presented above indicate that copolymers of U and G cont#ain a high dcgwe of srcondary st,ructure when the relative cont,cnt of G is high, and this interpretation is consistent with recent report#s on the secondary structure of poly (: itself.?`, 25, 28-30 (1) AMlough the four UG preparations described have approxi- mately the same chain lengths the sedimentat,ion coefficient of poly UG (OX/l j at neutral pH is markedly higher than that of the others. This increase in S value may result from greater aggregation (due to hydrogen bonding) with increasing G content for, at pH 12, where the secondary structure of poly G collapses,Z5f 28 all t,he polymers have similar sedimentation characteristics. (2) The sharp drop in phosphorolysis rat,e observed on going from a poly UG with a U/G ratio of 1.6'1 t(o one with a U/G ratio of 0.58/l can also be explained by an increase in secondary struct,urc. Ochoa3' and Grunberg-Manago3" have shown that polyribonucleotides haCng ordered secondary structures are phosphorolyzed much less rapidly t,han polymers existming as random coils. Poly G, for example, is completely resistant to phosphorolysis by polynucleot,ide phosphorylase.lg, 3Z (3) Only polymers contain- ing relatively large amounts of G (U/G, l&/l, and 0.58/l) showed any increase in ultraviolet absorption at raised temperatures, and the increase is greater the higher the G content. (4) The relatively high optical rotaCon of poly UG (U/G, 058/I) also indicates ordered secondary structure.4 Thus, the first t,hrce polynucleotides listed in Table 1 contain only moderate amounts of secondary struct'ure and direct amino acid into protein with high efi- cicncy. The last polymer, which contains almost 70 per cent G, exhibits a great deal of secondary structure and a markedly decreased ability to serve as a template for protein synt,hesis. This correlation between template efficiency and secondary struct,llre is consistent with our earlier observation that the ability of poly U to dir& polyphenylalaninc synthesis is lost when poly A-poly U helices are formed.g L'urt,hermore, we have foundlo that the extent of inhibition of polyphenylalanine synthesis by oligoadenylic acid preparabions increases with the length of the oligo- nuclcot,idc chain and can be correlated with the stability of oligoadenylic-poly U helices. Thus, single st'randedncss and lack of extensive intramolecular hydrogen bonding apptw t)o Iw wquisitc for messenger RPI;A activity in this in vitro system. Result,s obtained wit,11 natural TWA preparat,ions arc in accord with this conclusion. For 308 BIOCHEMIBTRY: SINGER ET AL. PROC. N. h. s. example, TMV-RKA directs protein synthesis wit.h high efficiencys, 34 in this sys- tem, compared to the efficiency of other RNA preparations.g Melting curves for T&IV-RNA4* 5 indicate that a larger per cent of its secondary structure is destroyed at 37' than is destroyed with ribosomal or transfer RNA preparat#ions. From an experimental point of view, caution should be used in handling templabe RNA to be used in in vitro systems. In the course of experiments on the melting of polymers we noted that the change in absorption with temperature is not always readily reversible. Thus the storage (for example, freezing and thawing) of poly- mer solutions may cause significant changes in secondary structure and therefore in templat'e efficiency. At present three factors influencing template activity are known-the size of the polymer chain,14z 15, 33, 35 the nucleotide sequence, and the secondary structure. Previously we showed that poly U fractions of relatively high molecular weight are more active as templates than smaller molecules14 and more recent dat'a indicate that high template activity corresponds to average chain lengths of 100 or greater.33* 35 The effect of nucleotide sequence on template efficiency is difficult to assess. Recent data show that the code is very degenerate15 but it is not known whether all nucleotide sequences will code. Although nonsense sequences may exist, thus far none have been definitively demonstrated. As indicated in the pres- ent report, the secondary structure of a polynucleotide must also be considered in any evaluation of its ability to serve as template RNA in the in vitro system. The molecular basis for the genetic information specifying the beginning or end of a protein is unknown. Previously we suggested that nonsense nucleotide sequences might function in this manner." The results of the present study suggest an al- ternative explanation. Thus, a limited region of messenger RNA containing a high degree of secondary structure as a result of intramolecular hydrogen bonding (for ex- ample, G-G interaction) might also specify the beginning or end of a protein. Areas of marked secondary structure, which may be likened to knots in a rope, might separate the messenger molecules containing information for the synthesis of more than one protein into functional units. Mechanisms enabling certain pro- teins to bc synthesized in close geographic proximity may be advantageous as, for example, in the synthesis of proteins containing different subunits36, 37 or for the syn- thesis of coordinately repressed enzymes. In a more general sense, we expect that future studies of the three distinct RNA fract'ions, transfer RNA, ribosomal RNA, and messenger RTU'A, will indicate functional significance for the specific conforma- tional aspects of their structures. With regard to messenger RNA, we would sug- gest that both the nucleotide sequence and secondary structure are relevant to t,he decipherment of the genetic code. Summary.-A series of copolymers containing varying amounts of uracil and guanine have been studied as templates in a cell-free system for protein synthesis. Polymers containing relatively large proportions of guanine have low template ac- tivity. Evidence indicating that the guanine-rich polymers contain a high degree of ordered secondary structure is presented. It is suggested that the existence of such secondary structure accounts for the poor template activity of these polymers. The possible functional significance of RKA secondary structure in the coding mechanism is discussed. VOL. 49, 1963 BIOCHEMISTRY: SINGER ET AL. 399 It is a pleasure to thank Linda Greenhouse and Helen Maleady for valuable technical assist- ance. We would also like to thank Dr. Samuel Luborsky for his experimental and theoretical a+ sistance with the sedimentation studies. * The following abbreviations are used: Poly U, polyuridylic acid; poly A, polyadenylic acid; poly C, polycytidylic acid; poly G, polyguanylic acid; poly UC, copolymer of uridylic and cytidylic acids; poly UC, copolymer of uridylic and guanylic acids; G, guanylic acid; U, uridylic acid; A, adenylic acid; C, cytidylic acid; TMV, tobacco mosaic virus. All other abbreviations conform to those acceptable to the Journal of Biological Chemistry. 1 Watson, J. D., and F. H. C. Crick, Nature, 171, 737 (1953). * Watson, J. D., and F. H. C. Crick, Nature, 171, 964 (1953). 3 Josse, J., A. D. Kaiser, and A. Kornberg, J. Biol. Chem., 236, 864 (1961). 4 Doty, P., H. Boedtker, J. R. Fresco, R. Haselkorn, and M. Litt, these PROCEEDINGS, 45, 482 (1959). 5 Doty, P., H. Boedtker, J. R. Fresco, B. D. Hall, and R. Haselkorn, Ann. 11'. Y. Acad. 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