in protein synthesis proceeds by an analogous mechanism, in which puromycin is

Transcription

1 ROLE OF GUANOSINE 5'-TRIPHOSPHATE IN THE INITIATION OF PEPTIDE SYNTHESIS, I. SYNTHESIS OF FORAI YLAIETHIONYL-PUROAI YCIN* BY J. W. B. HERSHEY AND R. E. THACH JOHN COLLINS WARREN LABORATORIES OF HUNTINGTON MEMORIAL HOSPITAL OF HARVARD UNIVERSITY AT MASSACHUSETTS GENERAL HOSPITAL, BOSTON, AND DEPARTMENT OF CHEMISTRY, HARVARD UNIVERSITY Communicated by Paul Doty, January 27, 1967 The reaction of puromycin with formylmethionyl-srna (F-met-sRNA) in the presence of ribosomes and ApUpG messenger to give formylmethionyl-puromycin (F-met-puro) has been described recently by -Marcker and Bretscherl and by Zamir et al.2 The results of their experiments suggest that in the course of this reaction F-met-sRNA binds first to the site on the ribosome normally occupied by peptidylsrna (the P site) and then reacts with puromycin, which is bound to the site normally occupied by aminoacyl-srna (the A site). This process is reported to require neither GTP nor supernatant enzymes.2 On the basis of these and other experiments3 4 it has been postulated that the formation of the initial peptide bond in protein synthesis proceeds by an analogous mechanism, in which puromycin is replaced by an aminoacyl-srna molecule. According to this model, the first peptide bond forms spontaneously (or is catalyzed by a ribosomal enzyme3) once the two species of aminoacyl-srna are bound, without the involvement of GTP. In order to test this model we have studied the conditions for dipeptide synthesis with the messenger ApUpGpUpUpU. In sharp contrast to the predictions of the theory, we have observed an absolute requirement for GTP in the formation of the dipeptide formylmethionyl-phenylalanine.5 Moreover, 5'-guanylyl-methylenediphosphonate (GMP-PCP), an analogue of GTP which is a strong competitive inhibitor of protein synthesis,' inhibits this reaction in the presence of GTP. This surprising result led to a reinvestigation of the reaction of puromycin with F-metsRNA described above. In particular, it was important to determine whether this reaction required the presence of GTP, under conditions optimal for dipeptide synthesis. Contrary to the results of others,2 our experiments show clearly that GTP strongly stimulates the formation of F-nmet-puro, and that GM\P-PCP inhibits this reaction. Moreover, it has been demonstrated that GTP acts in a step prior to the actual formation of F-met-puro, but subsequent to the binding of F-met-sRNA to the ribosome. Materials and Methods.-Ribonomes and S-30: S-30 was prepared from E. coli strain A19 or 1113 (a strain obtained from Dr. W. Gilbert, which lacks RNase I and polynucleotide phosphorylase and which has a temperature-sensitive RNase II) essentially according to the procedure of Capecchi and Gussin,7 except that the crude cell extract was made 1 mm in dithioerythritol before centrifuging at 30,000 X g. Ribosomes were prepared by the method of Ganoza.8 S-30 was centrifuged at 105,000 X g for 3 hr. The upper two thirds of the supernatant was removed, dialyzed against 0.01 M Tris-HCl, ph 7.5, and M glutathione, and stored in liquid nitrogen as the S-100 preparation. The ribosomal pellet was suspended gently in 0.01 M Tris-HCl, ph 7.6, M Mg(OAc)2, and 0.50 M NH4OAc and centrifuged 4 hr at 105,000 X g. The supernatant was saved as the source of initiation factors. The ribosomes were centrifuged twice more in a buffer containing 0.01 M Tris-HCl, ph 7.6, 0.01 M Mg(OAc)2, and 0.50 M NH4OAc. The final pellet was suspended in 759

2 760 BIOCHEMISTRY: HERSHEY AND THACH PROC. N. A. S M Tris-HCi, ph 7.7, and 0.01 M Mg(OAc)2 and clarified by 15 min centrifugation at 30,000 X g. The ribosomes were stored at a concentration of 20 mg/ml in ice and could be kept this way for 3-4 weeks with little loss of activity. At no time during the preparation were they frozen. Initiation factors: The supernatant from the first ribosomal wash was brought to 80% saturation with solid (NH4)2SO4, during which the ph1 was constantly adjusted to 7.8. The precipitate was dissolved in 0.01 M Tris-HCl, ph 7.8, 0.01 M Mg(OAc)2, and 0.06 M KCl and dialyzed overnight against the same buffer. The solution was passed through a 1 X 20 cm column of G-25 Sephadex equilibrated with 0.02 M sodium phosphate, ph 6.0. After 3 hr dialysis against the Tris-Mg-KCl buffer above, the factor preparation was divided into small aliquots and frozen. Formylated C'4-met-sRNA: E. coli B srna (Schwarz BioResearch, Inc.) was further purified according to Holley et al.9 by elution from a DEAE-cellulose column with 1 M NaCl in 0.01 M The srna was recovered by ethanol precipitation, dissolved in a small volume Tris-HCl, ph 7.5. of M KOAc, ph 5, and M glutathione, and dialyzed against water overnight. Two mg of srna were charged with C'4-methionine in a total volume of ml containing: 200 mm 1 Tris-HCl, ph 7.1, 20 mm, Mg(OAc), 10 mm glutathione, 7.5 mm ATP, 0.2 mm CTP, 20 mm creatine phosphate, 70 Mg/ml creatine phosphokinase (Sigma Chemical Co.), 0.6mM folinic acid (General Biochemicals), 0.03 mm each 19 cold amino acids (less Met), 6 Mucuries C14-methionine (New England Nuclear Corp., specific activity 187 mc/mmole), and 0.20ml/ml of S-100. After 20 min incubation at 370 the solution was cooled in ice, 20,moles EDTA was added, and the protein was extracted thoroughly with 1 ml phenol. The srna was then precipitated with ethanol, dissolved in 1 ml water, and passed through a 1 X 20 cm G-25 Sephadex column. The first peak, as determined by absorption at 260 mpa, was pooled and lyophylized. The charged srna was dissolved in water and M fl-mercaptoethanol, the ph was adjusted to 6, and the solution was stored frozen. The extent of charging varied from 3 to 4% on a molar basis, with roughly 70% of the methionine formylated. Puromycin reaction and assay: The concentration of ingredients in the complete reaction mixture was: 0.05 M Tris-HCl, ph 7.1, 0.10 M NH4OAc, 10 mm Mg(OAc)2 or as stated, 1 mm GTP or as stated, 0.25 mm ApUpG, 1 mm puromycin, 0.2 mm GMP-PCP (when added), 2 mg/ml ribosomes, 0.10 ml/ml initiation factors, and 0.285,M formylated C14-met-sRNA (53 m,4c/ml). The concentrations of ApUpG, ribosomes, and factors indicated were those which gave optimal rates of reaction. Incubations were at 200. The amount of F-met-puro formed per 20 Ml aliquot was measured by the method of Leder and Bursztyn.'0 We have confirmed by paper chromatography and electrophoresis that more than 95% of the radioactivity extracted into the ethylacetate phase migrates as F-met-puro. By inspection of the apparent first-order reaction kinetics, the maximum extent of reaction could be estimated and log plots of the data constructed. These plots gave straight lines with good fit, and the slopes of these lines were used as a measure of the relative rates of reaction. Essentially the same relative rates could be estimated from the observed initial rates of reaction, but the calculations using log plots, which involved all the time points, seemed more reliable. Binding assay: Reaction mixtures contained 0.05 M Tris-HCl, ph 7.1, 0.10 M NH4OAc, 0.01 M Mg(OAc)2, 0.25mM ApUpG, 2 mg/ml ribosomes, 0.1 ml/ml initiation factors, 1 mmi GTP when added, and MAM formylated C'4-met-sRNA (53 muc/ml). Incubation was at 20 ; aliquots (10 Ml) were assayed according to Nirenberg and Leder."1 The synthesis of ApUpG has been described by Sundararajan and Thach." The GTP inhibitor, GMP-PCP, was prepared as described by Hershey and Monro.6 Results.-Requirement for GTP in the formation of F-met-puro: To determine the effect of GTP on the rate of F-met-puro formation, reaction mixtures were prepared with and without added GTP, and with added GMP-PCP. The reaction protocol and assay were as described in Materials and Methods. The time course of F-met-puro formation is shown in Figure 1. It is clear that GTP strongly stimulates the rate of this reaction. i/ioreover, GMIP-PCP reduces the rate below that obtained in the absence of GTP, suggesting that the reaction mixtures may contain a trace of contaminating GTP (or a contaminating enzyme system which generates GTP). The dependence of the GTP-stimulated reaction upon MJg++ concentration

3 VOL. 57, 1967 BIOCHEMISTRY: HERSHEY AND THACH 761 is shown in Figure 2. It is apparent that the optimum Mlg++ concentration is around 10 mm, similar to that observed in the binding of F-met-sRNA to ribosomes12 and in the incorporation of F-met into polypeptide.13 This result suggests that the GTP-dependent synthesis of F-met-puro is physiologically significant, and not an artifact induced by abnormal conditions. Effect of freezing ribosomes on the GTP-stimulated reaction: Initial investigations of the GTP dependence of F-met-puro synthesis did not give the results shown in Figure 2. Instead, GTP dependence was observed only at very low Mg++ concentrations (around 4-5 mm). At 10 mm1\ig++, there was no stimulation by GTP, and no inhibition by G1\IP-PCP. Only when unfrozen ribosomes8 were used did the strong stimulation by GTP (see Fig. 2) become apparent. The rate obtained with these ribosomes in the absence of GTP was similar to that observed with previous preparations of ribosomes which had been frozen, suggesting that freezing per se may have inactivated the GTP-dependent reaction. It was then of interest to test directly the effect of freezing on the GTP-dependent reaction. Aliquots of previously unfrozen ribosomes were frozen in liquid nitrogen and quickly thawed at 200, four times and eight times. It is evident (Fig. 3) that freezing and thawing markedly. reduces the rate of the GTP-dependent reaction, whereas the rate of reaction in the absence of GTP is unaffected. This result suggests that the GTPdependent reaction is extremely labile, and may be inactivated by relatively mild manipulations. This may explain at least in part why other workers2 have not observed GTP dependence. Formation of F-met-puro in S-30 extracts: While freezing and thawing, and 10 cr 6_-o 0 F,-COMPLETE~~~~~~~~~~~~~~~~~~OPLT -Grp~ ~ ~ ~~~~ 0 -T TIME,MIN FIG. 1.-Rate of formation of F-met-puro. Mg++ conc., mm. Reaction conditions are described in Materials and Methods. Calculations of,opmoles FIG. 2.-Variation of relative rates with from cpm were based on the assumption that Mg++ concentration. Reaction conditions only 50% of the F-met-puro is extracted from and calculation of relative rates of formation the aqueous solution.'0 Symbols represent: of F-met-puro are described in Materials and complete system (s); without GTP (h); Methods. Symbols represent: complete sy~swithout GTP but with GMP-PCP (s); tern (s); without GTP (A); without GTP without ApUpG (XM). (-). but with GMP-PCP

4 762 BIOCHEMISTRKY: HERSHEY AND THACH Paoc. N. A. S. perhaps other purification procedures, seemed to depress the GTP-dependent reaction, it was important to determine whether GTP dependence itself might also be an artifact induced by purification. To test this possibility, the synthesis of F-met-puro was studied using a fresh undialyzed S-30 preparation (see Materials and Methods) as a source of ribosomes and initiation factors. The effects of GTP and GMP-PCP on this reaction are shown in Figure 4. It is clear that GTP strongly stimulates the formation of F-met-puro at early times; the subsequent decrease in the amount of F-met-puro seems most probably due to deformylation by an enzyme which is known to be present in crude extracts.14 GMP-PCP depresses the rate somewhat below that obtained in the absence of GTP, again suggesting a low level of GTP contamination. These results indicate that the GTP dependence of this reaction is not an artifact caused by purification of ribosomes or factors. Effect of GTP on the binding of F-inet-sRNA (unfractionated) to ribosoines: It was now of interest to determine more precisely the mechanism by which GTP enhances the rate of F-met-puro synthesis. Since the first step in this reaction is thought to be the binding of F-met-sRNA to the ribosome, the effect of GTP on the rate of this reaction was studied. A typical result is shown in Figure 5, from which it is evident that GTP has only a small effect on the rate of binding. Although this result was obtained using unfractionated srna, it seems unlikely that a large proportion of the radioactivity bound is due to met-srnam;15 over 55 per cent of the total radioactivity added was bound, indicating that at the very z 10 8 UNFROZEN 10 I 0 ~~~~~~~4 xfrozen 8-0 < ~~~~~~~~~~~~~~~~~~~~I-- Cr< 7E 8xFRWEN X 6- w 4 0 J4- GTp TIME, MIN TIME, MIN. FIG. 3.-Effect of freezing ribosoines on the GTP-stimulated reaction. Small portions of the same preparation of ribosomes were frozen and thawed as indicated and then used in the puromycin reaction as in Fig. 1. The open symbols show the amount of F-met-puro formed in the presence of 1 mm GTP after the ribosomes were: unfrozen (0); 4 times frozen (A); 8 times frozen ( Ei). Closed symbols represent the corresponding reactions without GTP. FIG. 4.-Formation of F-metpuro in S-30 extracts. Reaction conditions were identical to those in Fig. 1, except that the ribosomes and initiation factors were replaced by 0.2 ml/ml S-30. Symbols represent: complete system (0); without GTP (A); without GTP but with GMP-PCP (o); without ApUpG (X).

5 VOL. 57, 1967 BIOCHEMISTRY: HERSHEY AND THACH 763 most, less than half of this could be due to metsrnam. Moreover, it is probable that the pro- COMLETE portion of met-srnam bound is much lower z 12,c than this upper limit, since the binding of this /v w species is very poor at 0.01 M g++."5 Recently, this experiment has been repeated,f using purified F-met-sRNAF, with a similar re- '4 sult. Moreover, it has been shown that the F-met-sRNAF prebound to ribosomes in the ab- sence of GTP (in which state it is unreactive 0 10 L TIME, MIN. with puromyciin; see below) can subsequently be activated by the addition of GTP, with the FIG. 5.-Rate of binding of F-mets1RNA to ribosomes. Reactioin coinresult that it now reacts rapidly with puro- ditionis are described inl Materials and mycin. These experiments with purified F-met- Methods. Symbols represent: complete system (0); without GTP srnaf will be reported elsewhere. (a); Thus it may without ApUpG (o). be tentatively concluded that GTP affects slightly, if at all, the rate of binding of F-met-sRNAF to ribosomes. This implies that the GTP-dependent reaction occurs subsequent to the binding step. A further investigation of this point is in progress. Preincubation with GTP in the absence of puromycin: Having obtained evidence that GTP is probably involved in a step subsequent to the binding step, we next sought to determine whether the presence of puromycin was required for the action of GTP. To test this possibility, experiments were conducted in which the ribosomes, charged srna, and factor were preincubated in the presence or absence of GTP prior to the addition of puromycin. The results of these experiments are shown in Figure 6. First, it is evident that preincubation without GTP, for a time sufficient to allow near-maximal binding of srna to ribosomes (8 min), had little or no effect on the subsequent rate of F-met-puro synthesis (puromycin and GTP were added simultaneously after preincubation). In contrast, inclusion of GTP in the preincubation mixture resulted in a very fast spurt of F-met-puro synthesis when puromycin was added, the extent of this rapid incorporation being greater the longer the preincubation. This result indicates that the GTP reaction proceeds in the absence of puromycin, and gives rise to an activated complex which then reacts extremely fast with added puromycin. The time course of this GTP reaction in the absence of puromycin can be obtained by plotting the magnitude of the initial spurt as a function of preincubation time. Although not shown here, this time course is superimposable on that obtained without preincubation (lower curve, Fig. 6), indicating that the GTP reaction is rate-determining in the absence of preincubation and that this rate is unaffected by the presence of puromycin. A further indication that the step involving GTP occurs prior to and independent of the actual reaction of puromycin with F-met-sRNA (that is, the formation of the amide bond) is obtained from experiments in which GMP-PCP was added after preincubation with GTP but before the addition of puromycin. If the GTP reaction were essentially completed during the preincubation period, then the subsequent addition of GMAIP-PCP should not affect the magnitude of the spurt of F-met-puro formed upon the addition of puromycin. That this is the case is

6 764 BIOCHEMISTRY: HERSHEY AND THACH PROC. N. A. S. evident from Figure 7A. The addition of GMP-PCP after preincubation with GTP does not alter the height of the spurt (obtained by extrapolation to zero time), whereas inclusion of GMP-PCP during preincubation reduces it considerably. A control reaction (Fig. 7B) shows the inhibitory effect of GMIP-PCP in the absence of preincubation. Discussion.-The results presented above demonstrate that GTP is required for the formation of F-met-puro, that this reaction shows the same optimum Mg++ concentration as the initiation reaction in protein synthesis,", 13, 16 and that it is not an artifact caused by purification. Moreover, evidence has been obtained which suggests that the reaction involving GTP occurs subsequent to the binding of F-met-sRNA to ribosomes but prior to the actual formation of the amide bond between formylmethionine and puromycin. There are several model reaction mechanisms which can account for the above 0 TIME, MIN. FIG. 6.-Preincubations in the absence of puromycin. The solid lines show the extent of formation of F-met-puro under a variety of conditions of preincubationl. The final concentrations of components in the reaction mixtures are the same as those described in Materials and Methods. The open symbols represent preincubations in the presence of GTP for the -following times: 8 mmn (0); 4 mi (A); 2 min (n); 1lmin (V), The closed symbols represent the corresponding reactions in which GTP was omitted during preincubation, but added later at zero time with the puromycin. The symbol (X) represents no preincubation at all. The dashed lines are extrapolations to zero time and can be used to estimate the extent of the fast spurt of F- met-puro formation. TIME, MIN. FIG. 7.-Lack of inhibition by (GMP-PCP of spurt of F-met-puro formation. Reaction conditions are similar to those in Fig. 6, except the GTP concentration was 0.3 mm in all the incubations, and the GMP-PCP concentration was 2 mm whenever added. Preincubations were 20 mi and purom cin was added at zero time. The figure shows the extent of formation of F-met-puro with time. (A) Symbols represent: preincubation without GMP-PCP, then only puromycin added at zero time (a); preincubation without GMP- -PCP, but (MP-PCP and puromycin added at zero time (n); preincubation with GMP- PCP, then puromycin added at zero time (A). (B) Controls. The rate of formation of F-met-puro without any preincubation is shown in the presence (o) and in the absence (0) of GMP-PCP.

7 VOL. 57, 1967 BIOCHEMISTRY: HERSHEY AND THACH 765 facts. Of these we favor the following, which may be termed the "single entry site model." The salient feature of this scheme is the postulate that with natural, undamaged ribosomes, the F-met-sRNA first binds to the A site, and not to the P site, as previously supposed.1' 2 This binding probably does not involve the supernatant factors4 or GTP, but may well require one or both initiation factors."7 18 Indeed, preliminary results obtained in our laboratory19 show a strong stimulation of F-met-sRNA binding, as well as F-met-puro formation, by the addition of partially purified initiation factors. This result confirms the findings of Salas et al.18 After this initial binding at the A site, we propose that GTP is involved in a transfer step which moves the F-met-sRNA from the A to the P site. This step is completely analogous to the transfer step postulated by Traut and M\onro20 in the case of bound polyphenylalanyl-srna. It probably requires one or more of the supernatant factors,4 particularly the one associated with GTPase activity. Once the F-met-sRNA has moved to the P site, the A site is available for the binding of puromycin or aminoacyl-srna, which then reacts with the adjacent F-met- srna to give either F-met-puro or formylmethionyl-aminoacyl-srna, respectively. It should be noted that this model is similar to that proposed by Heinz and Schweet2' for initiation in the rabbit reticulocyte system. While this mechanism is in accord with the results presented above, it does not account for the low level of F-met-puro formation observed in the absence of GTP nor for the result obtained with frozen (damaged) ribosomes, where there is no detectable effect either of GTP or of GMP-PCP on the rate of reaction at 10 mm MIg++. These observations suggest the possibility that under certain conditions F-met-sRNA can bind directly to the P site without the involvement of GTP, albeit at a very low rate. Further work is required to verify this point. However, whatever may be the explanation for these discrepancies, it seems clear that the physiologically significant mechanism in F-met-puro synthesis requires GTP. One of the major conceptual difficulties raised by this model is that it does not explain how a single codon, AUG, can code for two different amino acids (methionine and formylmethionine) without confusion. As we have previously shown,22 in the initiation of polypeptide synthesis AUG is read exclusively as a formylmethionine codon; no free methionine is incorporated. At presentwecan onlyspeculate as to how this distinction might be accomplished. One possibility is that the initiation factors alter the specificity of the A site such that only F-met-sRNA can be bound. Alternatively, the specificity of the A site may depend on whether the adjacent P site is occupied by a peptidyl-srna. If the P site is occupied, then the A site might accept only aminoacyl-srna's; if it is vacant, then the A site might be altered such that it would accept only F-met-sRNA. We are currently investigating several of these possibilities. A second general model which would account for the GTP dependence of F-metpuro synthesis involves the postulate that GTP activates bound F-met-sRNA by some means other than the site transfer mechanism. This activation would probably involve the hydrolysis of GTP, rather than an allosteric interaction, since G'i\IP-PCP (which presumably cannot be hydrolyzed6) is a strong competitive inhibitor of the reaction. This "chemical activation" model would allow the direct binding of F-met-sRNA to the P site. This is one of the most important features of previous models1' 12 as it affords a good explanation of how AUG can code

8 ,-66 BIOCHEMISTRY: HERSHEY AND THACH PROC. N. A. S. unambiguously for formylmethionine in initiation (the basic proposal is that F- met-srna can bind only to the P site, whereas met-srna can bind initially only to the A site). The major difficulty with the chemical activation model is that it requires the existence of two very different types of reactions involving GTP: (a) the activation of F-met-sRNA in initiation and (b) the normal site transfer reaction in the process of chain elongation.20 However, more complicated variations of this model can be devised to eliminate this problem. Finally, attention should be drawn to the proposal of Seeds and Conway23 that a GTP-dependent reaction expels uncharged srna from one or more sites on the ribosome. It seems possible that the ribosomes as prepared above contain ull- (harged srina which blocks either or both binding sites. Hence, GTP may be required to clear these sites before the reaction between F-met-sRNA and puromyciin can take place. Summary.-The reaction between F-met-sRNA and puromycin in the presence of ribosomes, initiation factors, and ApUpG to form F-met-puro requires GTP, and is inhibited by GMPIP-PCP. We are indebted to Miss Nadia Mykolajewycz and Mrs. K. F. Dewey for expert technical assistance. We also wish to thank Drs. Paul Doty and P. C. Zamecnik for advice and encouragement during the course of these experiments and in the preparation of the manuscript. * Supported by research grants from the American Cancer Society, Inc. (E-170A and E-51) and from the National Institutes of Health (HD-01229). This is publication No of the Cancer Commission of Harvard University. 1 Bretscher, M. S., and K. A. Marcker, Nature, 211, 380 (1966). 2 Zamir, A., P. Leder, and D. Elson, these PROCEEDINGS, 56, 1794 (1966). 3 Maden, B. E. H., R. R. Traut, and R. E. Monro, in Abstracts, Federation of European Biochemical Societies, Third Meeting, Warsaw (1966), p Nishizuka, Y., and F. Lipmann, Arch. Biochem. Biophys., 116, 344 (1966). 5 Thach, R. E., and K. F. Dewey, manuscript in preparation. 6 Hershey, J. W. B., and R. E. Monro, J. Mol. Biol., 18, 68 (1966). 7 Capecchi, M. R., and G. Gussin, Science, 149, 417 (1965). 8 Ganoza, M. C., private communication. 9 Holley, R. W., J. Apgar, B. P. Doctor, J. Farrow, M. Marini, and S. Merrill, J. Biol. Chem., 236, 200 (1961). 10 Leder, P., and H. Bursztyn, Bwchem. Biophys. Res. Commun., 25, 233 (1966). 11 Nirenberg, M. W., and P. Leder, Science, 145, 1399 (1964). 12 Sundararajan, T. A., and R. E. Thach, J. Mol. Biol., 19, 74 (1966). 13 Brown, J. C., and It. E. Thach, unpublished experiments. 14 Capecchi, M. R., these PROCEEDINGS, 55, 1517 (1966). 15 Clark, B. F. C., and K. A. Marcker, J. Mol. Biol., 17, 394 (1966). 16 Kolakofsky, 1)., and T. Nakamoto, these PROCEEDINGS, 56, 1786 (1966). 17 Stanley, W. M., Jr., AI. Salas, A. J. Wahba, and S. Ochoa, these PIROCEEDINNGs, 56, 290 (1966). 18 Salas, M\., MI. B. Hille, J. A. Last, A. J. Wahba, and S. Ochoa, these PROCEEDINGS, 57, 387 (1967). 19 Sarkar, S., and R. E. Thach, unpublished results. 20 Traut, R. R., and R. E. Monro, J. Mol. Biol., 10, 63 (1964). 21 Heinz, R., and R. Schweet, in Cold Spring Harbor Symposium on Quantitative Biology, vol. 31, in press. 22 Thach, R. E., K. F. Dewey, J. C. Brown, and P. Doty, Science, 153, 416 (1966). 23Seeds, N. W.. and T. W. Conway, Biochem. Biophys. Res. Commun., 23, 111 (1966).