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Home , Alanine (data page), Amino acid replacement, Asparagine, Asparagine (data page), Glycine (data page), Leucine

VOL. 48, 1962 : E. L. SAIITH 677

18 Britten, R. J., and R. B. Roberts, Science, 131, 32 (1960). '9 Crestfield, A. M., K. C. Smith, and F. WV. Allen, J. Biol. Chem., 216, 185 (1955). 20 Gamow, G., Nature, 173, 318 (1954). 21 Brenner, S., these PROCEEDINGS, 43, 687 (1957). 22 Nirenberg, M. WV., J. H. Matthaei, and 0. WV. Jones, unpublished data. 23 Crick, F. H. C., L. Barnett, S. Brenner, and R. J. Watts-Tobin, Nature, 192, 1227 (1961). 24 Levene, P. A., and R. S. Tipson, J. Biol. Ch-nn., 111, 313 (1935). 25 Gierer, A., and K. W. Mundry, Nature, 182, 1437 (1958). 2' Tsugita, A., and H. Fraenkel-Conrat, J. Mllot. Biol., in press. 27 Tsugita, A., and H. Fraenkel-Conrat, personal communication. 28 Wittmann, H. G., Naturwissenschaften, 48, 729 (1961). 29 Freese, E., in Structure and Function of Genetic Elements, Brookhaven Symposia in Biology, no. 12 (1959), p. 63.

NUCLEOTIDE CODING AND AM1INO REPLACEMIENTS IN * BY EMIL L. SMITHt

LABORATORY FOR STUDY OF HEREDITARY AND METABOLIC DISORDERS AND THE DEPARTMENTS OF BIOLOGICAL CHEMISTRY AND MEDICINE, UNIVERSITY OF UTAH COLLEGE OF MEDICINE Communicated by Severo Ochoa, February 14, 1962 The problem of which bases of messenger or template RNA' specify the coding of amino in proteins has been largely elucidated by the use of synthetic polyri- bonucleotides.2-7 For these triplet nucleotide compositions (Table 1), it is of in- terest to examine some of the presently known cases of substitutions in polypeptides or proteins of known structure. The code appears to be universal, that is, it is the same in all species.6 It is assumed that a involving the substitution of one amino acid for another in a of the same species, e.g., human hemoglobin, represents an alteration in the triplet code in which only a single base of the three is replaced without al- teration of the sequence of the other two bases. A change of two or more bases is less likely. Moreover, in those cases for which the code for two amino acids is represented by the same triplet composition of bases, a substitution by an amino acid possessing the same code composition would be unlikely, since this would amount to a double base substitution in order to accomplish the necessary trans- position in sequence. Various considerations suggest that the triplet code is of the nonoverlapping type; this has recently been discussed rather fully by Crick et al.,8 who have also reported evidence that a triplet code is involved. At the time this present evaluation was undertaken, the codes for only 14 amino acids were known.9 It was of interest to determine whether it was possible to pre- dict, on the basis of known amino acid substitutions, the codes for other amino acids. This proved to be valid for certain amino acids where sufficient information was available. This is illustrated below for , , . and . These data, as well as other, thus serve as a verification of the thesis that simple involving an amino acid substitution involve a change in a single nucleotide base of a triplet without alteration of the sequence in the triplet. Downloaded by guest on September 25, 2021 678 BIOCHEMISTRY: E. L. SMITH PROC. N. A. S.

TABLE 1 TRIPLET CODE LETTERS FOR AMINO ACIDS* Amino acid Code Amino acid Code Amino acid Code Alanine UCG UG2 UC2 UCG UAC U2C Asparagine UA2 (UAC)t U2A UAC (UC2) Aspartic acid UAG U2C (U2A, U2G) UG2 U2G UA2 U2A Glutamic acid UAG UAG U2G UCG UUU ...... * This table has been adapted from Speyer, Lengyel, Basilio, and Ochoa.6 These codes are, in general, similar to the 15 recently reported by Martin, Matthaei, Jones, and Nirenberg.7 The code for glutamine is not directly available. It was deduced from an in HNO2 mut ant of tobacco mosaic virus.4 t Code letters in parentheses represent additional letters for the amino acid (degenerate code).6 On the basis of this concept, it is useful to examine some homologous proteins of different species in which substitution of amino acids is known; this offers the pos- sibility of determining whether the alteration has involved a change in a single base or whether two or more base substitutions have occurred. Hence, it can be de- duced, in some instances, whether single or multiple steps in mutation have occurred during the evolution of species-specific proteins. Thus, a definition is at hand for direct permissible substitutions (single base change in the triplet) and for changes which would appear to be nonpermissible by a change in a single base. It is also of interest to examine briefly some amino acid substitutions as related to the known codes in terms of possible effects on the functions of proteins. Deduced Codes for Amino Acids.-From known amino acid substitutions, it was possible to deduce certain codes by making use of the code information then avail- able. Code for glutamic acid: In Table 2, there are listed the presently reported amino TABLE 2 SOME AMINO ACID REPLACEMENTS IN HUMAN HEMOGLOBINS Mutant ,_ __Mutant HbA Amino acid Type of Hb HbA Amino acid Type of Hb Glutamic acid Valine S10, 13 Lysine Aspartic acid F18 Glutamic acid Lysine C", E'2 Histidine Tyrosine MBostOn MEmory'5 Glutamic acid Glycine GSan Jose13 Histidine Arginine Zurich'5 Glutamic acid Glutamine GHonolulu14 Glutamic acid Alanine A220 Valine Glutamic acid MMilwaukeel' Serine Threonine A220 Asparagine Lysine Gphiladelphia.6 Threonine Asparagine A220 acid Glycine Aspartic Norfolk17 ...... acid substitutions in human hemoglobin (Hb). For glutamic acid, it is apparent that this amino acid has been replaced by lysine, valine, glycine, and glutamine. By assuming that only a single base of the triplet can be replaced, the code for glu- tamic acid can be deduced. Since lysine is UA2, the code for glutamic acid must contain at least one A. Since the valine code is U2G, the code for glutamic acid must also contain one U. The code for glycine is UG2; hence, the glutamic acid code must contain, in addition, one G. Therefore, the complete code for glutamic acid is UAG. This is consistent with the code for glutamine, UGC, which can be derived from the code for glutamic acid by a single base change. It is noteworthy that a mutant involving the change glycine to glutamic acid has been found also in the A protein of the tryptophan synthetase of E. coli.21 Code for aspartic acid: Inspection of Table 2 shows that aspartic acid has re- placed lysine (UA2) and glycine (UG2). In order for the code for aspartic acid to Downloaded by guest on September 25, 2021 VOL. 48, 1962 BIOCHEMISTRY: E. L. SMITH 679

have two bases in common with the codes for glycine and lysine, the code for as- partic acid must be UAG. Codefor asparagine: Asparagine has replaced threonine (UC2) in HbA2, and lysine (UA2) has replaced asparagine in HbGPhiladelphia (Table 2). This suggests that the code for asparagine is UAC. Code for alanine: The only known substitution for alanine in the hemoglobin series is in HbA2 in which it has replaced glutamic acid (UAG) although this may not be a simple mutation. Although such assumptions are somewhat risky, one may consider the known replacements for alanine in the homologous proteins of various species. The data of Pal6us and Tuppy,22 for the cytochromes c of various species, show that glutamine (UCG) and leucine (U2C) replace alanine and, in another position, that serine (U2C), glutamic acid (UAG), -and leucine (U2C) oc- cupy the same position as alanine. Also, in the of various species threonine (UC2) and alanine occur in the same position.23' 24 The codes for almost all of the aforementioned amino acids, viz., threonine, serine, leucine, and glutamine, con- tain U and C. Although some of the transformations for the triplet codes of these amino acids directly to one another and to alanine may involve more than one base change, it appears very likely that at least some of these are permissible changes, that is, that they involve only a single base change. The foregoing information suggests that the code for alanine would have to contain U and C in order to have two bases in common with the code for most of these amino acids. Since the codes for glutamic acid and glutamine both contain G, it is likely that the code for alanine is UCG. This has subsequently been shown to be the code for alanine.6 The Role of Uracil.-The most striking feature of the triplet base compositions for all of the amino acids thus far is that each triplet contains at least one U. Inas- much as the presence of U is the only unique feature of the base composition of RNA, as compared to DNA, an important and specific function for U in template or messenger RNA is implied. On the basis of a triplet code with 4 bases, there are 64 possible combinations. If, however, each code for an amino acid must contain one U, then all of the 27 possible codes lacking one U are excluded. This leaves for the 20 amino acids commonly found in proteins 37 potential triplets. The presently available information on mutants suggests that there are probably preferred positions in the triplet for the unique U although the sequences in the triplet are unknown. It is likely, for example, that there are 16 codes for amino acids in which U occupies the same unknown position of the triplet. This would represent all the possible codes for U in one position. This view is suggested by the following considerations. Again, we must suppose that a mutant involves a replace- ment of only a single base without alteration of sequence. Let us arbitrarily assume that U occupies the first position as a fixed one in the triplets for the following amino acids: glutamic acid (UAG), lysine (UA2), valine (U2G), glycine (UG2), and glutamine (UCG). Since the four last named amino acids can replace glutamic acid as a result of only a single base change, a U must occupy the same position in all five codes. Further, aspartic acid (UA G)and aspara- gine (UAC) can replace lysine (UA2), and asparagine can replace threonine (UC2); hence, in the codes for these three additional amino acids U appears to occupy the same position also. The same considerations also apply to serine (U2C), which can replace threonine. Arginine (UCG) is known to replace glycine (UG2) in trypto- Downloaded by guest on September 25, 2021 680 BIOCHEMISTRY: E. L. SMITH PROC. N. A. S.

phan synthetase (Table 3) and to replace histidine (UAC) in hemoglobin. Tyro- sine (U2A) can also replace histidine in hemoglobin. Thus, we can relate all of these codes with U in the same position for the aforementioned amino acids. Pheflyl- alanine (UUU) can obviously be included, making 13 codes thus far. Information concerning mutants in tobacco mosaic virus (see the summary by Speyer et al.6) suggests that isoleucine (U2A), leucine (U2C), and proline (UC2) complete the 16 possible triplet codes with U in the same position. The above tentative group ac- counts for all but four of the commonly occurring amino acids in proteins. Among the remaining four amino acids are tryptophan (UG2), which must have U in a different position from glycine, also UG2, and methionine (UAG), which must differ in its code for the position of U since the codes for glutamic acid and aspartic acid are also UAG. Similarly, since alanine, glutamine, and arginine all have the code UCG, one of these must have U in a different position than in those mentioned above. In view of the relationship of glutamine to glutamic acid and glycine to arginine, alanine may be the one which differs in the position of U. The code for cysteine completes the list but the relationships of its code to others are still un- known. From the above interrelationships it is apparent that when the triplet code se- quence is established for one amino acid, where three different bases are involved, it will be possible to deduce the sequence for essentially all of the other amino acid codes. Note added in proof: The work of Henning and Yanofsky2l on recombination of two mutants of E. coli to restore the normal A protein of tryptophan synthe- tase indicates that if the glutamic acid code is UAG, that for arginine is UGC. The G in each of these two amino acid codes nmust be in a different position to obtain by recombination UGG the code for glycine. This information together with that for the hemoglobin mutants should -facilitate absolute assignment of base sequences in the triplet codes. If in 16 amino acid codes U occupies the same position, as indicated above, one may assume that in the four other codes U occupies the same position, this is dif- ferent from the position for the 16 related codes. This suggests that there may be one position of U in the triplet which is nonfunctional or stated in the converse manner, each functional triplet must have U in one of two possible positions. Thus, of the 37 possible triplet codes containing U it would appear that only 28 may be functional. This hypothesis might aid in explaining the limited number of func- tional codes. It may be noted that Speyer et al.6 suggest that asparagine may be coded by UA2 and UAC on the basis of incorporation experiments. Inasmuch as lysine (UA2) pan replace asparagine in human hemoglobin, this transformation would involve the UAC code for asparagine. A transformation of asparagine as UA2 to lysine as UA2 would involve an unlikely double base change. Similarly, Speyer et al.6 suggest additional (degenerate) codes for leucine and threonine. The reported replacements involving threonine (Table 2) can be explained with UC2 as the code for this amino acid, but they can also be explained thus: serine (U2C) -- threonine (UAC) asparagine (.UA2). Relationships among Amino Acid Code Letters.-It is of interest to compare the Downloaded by guest on September 25, 2021 VOL. 48, 1962 BIOCHEMISTRY: E. L. SMITH 681

codes for certain amino acids which have functional properties in common. We may consider first those amino acids which possess large hydrophobic side chains and which presumably have similar or closely related properties within a protein molecule, i.e., where substitution of one of these amino acids for another would be expected to produce minimal disturbance in protein conformation. The code for phenylalanine is U3, for leucine U2C (U2A, U2G), for tyrosine U2A, for valine U2G, for isoleucine U2A, and for tryptophan UG2. It is noteworthy that a single base change would permit substitution of leucine, tyrosine, valine, or isoleucine for phenylalanine, whereas it is possible to derive the code for typtophan only from that of valine or leucine (U2G) among this group of amino acids, but not from any of the others. The code for methionine suggests that it too may be derived from that of valine and possibly some of the other hydrophobic amino acids. The code for cys- teine (U2G), as well as serine (U2C), may also be derived from the code for phenyl- alanine by a single base change. A few substitutions among hydrophobic residues are presently known within the homologous proteins of several species (Table 3). Unfortunately, such sub- stitutions are not known as yet within the same species and these are not likely to be easily found since the techniques presently used have largely depended on dif- ferences in electrophoretic mobility and hence in the charge of the proteins or the derived from them, e.g., as in the human hemoglobins. TABLE 3 SOME AMINO ACID REPLACEMENTS IN PROTEINS* Protein Species Amino acid Species Amino acid Reference Cytochrome c Mammalian Valine R. rubrum Phenylalanine 22 Cytochrome c Equine Threonine Bovine Serine 25 Bovine sheep Valine Horse, human, Isoleucine 23, 24 pig, etc. -y-Globulins Bovine, rabbit Phenylalanine Human Tyrosine 26 Tryptophan E. coli Glycine E. coli Glutamic acid 21 Synthetase A ..... Glycine .... Arginine * These are in addition to those mentioned in the text. The codes for the two dicarboxylic acids and their present certain interest- ing features. The codes for glutamic acid and aspartic acid prove to be both UAG. Hence, a mutation involving a single base change would not permit direct substitu- tion of one for the other. Nevertheless, such substitution is apparent in the reported structures for ACTH of several species.27 In human hemo- , substitution of glutamine for glutamic acid occurs. This substitution has: also been found in the y-globulins of different species.26 Conversion of the code for asparagine to glutamine by a single base change would appear to be possible, and this is known to occur in the derived from the 'y-globulins of different species.26 Thus, conversion of the code by a single base change from either glutamic acid or aspartic acid to asparagine is possible, but it cannot be possible for both or a double substitution would be involved. There is no present evidence as to which is the likely possibility, in the absence of sequence information for the triplet. The complex relationships among the codes for the dicarboxylic acids and their amides emphasizes the importance of determining the correct assignments in a polypeptide sequence. Substitution involving the two hydroxy amino acids, serine and thrconine, is Downloaded by guest on September 25, 2021 682 BIOCHEMISTRY: E. L. SMITH PROC. N. A. S.

known in a number of cases (Tables 2 and 3) which are intra- as well as inter- specific. In the insulins of various species, threonine (UC2, UAC) and alanine (UCG) occur in the same position. As noted above (see code of alanine), this could occur via a single base change in the triplet codes for these two amino acids. There is also known in the insulins a serine (U2C)-glycine (UG2) substitution. Clearly, on the basis of the triplet codes for these two amino acids, such a substitution is impossible by a mutation involving only a single base in the triplets. There are some features of the triplet codes which are surprising in terms of pro- tein chemistry. An aspartic acid-glutamic acid direct substitution by a single base change is, as already mentioned, not possible despite the similar properties of the two amino acids. The same is true for arginine and lysine, although function is not greatly altered in known cases of such substitution-cytochromes c,22 vasopres- sins,28 and ,3-melanophore-stimulating hormones.29 One possibility is that arginine (UGC)-lysine (UAA) replacement has occurred via an intermediate, such as histi- dine, whose code (UAC) has two bases in common with the codes for both arginine and lysine. An arginine-histidine substitution has been reported for human hemo- globin (Table 2). The properties of histidine are such that it may serve functionally in some circumstances in place of arginine or lysine, although the histidine analog of the is only weakly active.28 Amino acid substitutions in tobacco mosaic virus in relationship to the triplet codes have already been discussed.6 The substitution of arginine for glycine in a mutant of E. coli (Table 3) is of special interest in view of the profound difference in the properties of these two amino acids. Clearly, we are faced with several different problems in the evolution of . One of these is the preservation of function, in which we may envisage substitution in the polypeptide sequence of compatible amino acid residues whose side chain properties are similar and critical for enzymic or other functions. When replacements occurred involving amino acids of radically different properties and these proteins survived, evidently either the substitutions occurred at loci which were not critical for the function of the protein or new functions evolved when "active sites" were modified. Presently known cases suggest that in the hemoglobins, insulins, cytochromes c, and other proteins, many changes occurred at noncritical sites. In contrast to the above, we can visualize possible changes in function by amino acid substitution, as exemplified by the polypeptide hormones of the neurohypo- physis. The primitive weakly active of the frog30 (Table 4) could have TABLE 4 HORMONES OF THE NEUROHYPOPHYSIS Position 1 2 3 4 5 6 7 8 9 Vasotocin* CyS-Tyr-Ileu-Glu-NH2-Asp-NH2*CyS-Pro-Arg-Gly-NH2 CyS-Tyr-Phe Glu-NH2 Asp-NH2,CyS-Pro Arg*Gly-NH2 CyS-Tyr-Ileu-Glu-NH2-Asp-NH2-CyS-Pro-Leu-Gly-Nh2 * The half-cystine residues in positions 1 and 6 are in each case linked by a bridge. evolved into both vasopressin and oxytocin. The change from vasotocin to ar- ginine-vasopressin involves a change in position 3 from U2A (isoleucine) to U3 Downloaded by guest on September 25, 2021 VOL. 48, 1962 BIOCHEMISTRY: E. L. SMITH 683

(phenylalanine), only a single compatible base change in the triplet code. Like- wise, from vasotocin to oxytocin, the change in position 8 is from UCG (arginine) to U2C or U2G (leucine) which also represents a change in only a single base of the triplet code. It is of special interest that the possesses all three compounds in the neurohypophysis.31 For the evolution from a single hormone to the presence of two or more hormones in the same species, we must also assume a multiplication of the genetic material in order to have independent production of two or more hormones. The above case suggests that the investigation of the amino acid sequences of homologous proteins of various species should contribute much to our understanding of the evolution of function in the polypeptides and proteins. Summary.-Known amino acid replacements in mutants of human hemoglobin are consistent with a single base change in the known triplet codes. Certain amino acid replacements in homologous proteins of various species also involve single base changes whereas others do not; the latter probably involve stepwise multiple mutation. The data suggest that for 16 amino acids, uracil occupies the same but unknown position in the codes; the four other codes must involve a different loca- tion of uracil in the triplet. Problems of the maintenance and evolution of and protein structure and function are discussed briefly. * Aided by grants from the National Institutes of Health, U. S. Public Health Service. t Present address: University of Utah College of Medicine, Salt Lake City, Utah. 1 Abbreviations: RNA, ribonucleic acid; the capital letters U, A, C, and G are used for the nucleotide residues present in RNA, uridylic, adenylic, cytidylic, and guanylic, respectively. 2 Nirenberg, M. W., and J. H. Matthaei, these PROCEEDINGS, 47, 1558 (1961). 3 Lengyel, P., J. F. Speyer, and S. Ochoa, these PROCEEDINGS, 47, 1936 (1961). 4Speyer, J. F., P. Lengyel, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 63 (1962). 5 Lengyel, P., J. F. Speyer, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 282 (1962). 6 Speyer, J. F., P. Lengyel, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 441 (1962). 7Martin, R. G., J. H. Matthaei, 0. W. Jones, and M. W. Nirenberg, Biochem. Biophys. Res. Comm., 6, 410 (1962). 8 Crick, F. H. C., L. Barnett, S. Brenner, and R. J. Watts-Tobin, Nature, 192, 1227 (1961). 9 Thanks are due to S. Ochoa and J. F. Speyer for their courtesy in making available their results to us before publication. 10 Hunt, J. A., and V. M. Ingram, Nature, 184, 640 (1959). 11 Ibid., 181, 1062 (1958). 12 Ibid., 184, 870 (1959). 13 Hill, R. L., R. T. Swenson, and H. C. Schwartz, J. Biol. Chem., 235, 3182 (1960). 14 Swenson, R. T., R. L. Hill, H. Lehmann, and R. T. S. Jim, J. Biol. Chem., in press. 15 Gerald, P. S., and M. L. Efron, these PROCEEDINGS, 47, 1758 (1961). 16 Baglioni, C., and V. M. Ingram, Biochim. Biophys. Acta, 48, 253 (1961). 17 Baglioni, C., J. Biol. Chem., 237, 69 (1962). 18 Murayama, M., Federation Proc., 19, 78 (1960). 19 Muller, C. J., and S. Kingma, Biochim. Biophys. Acta, 50, 595 (1961). 20 Ingram, V. M., and A. 0. W. Stretton, Nature, 190, 1079 (1961). 21 Helinski, D. R., and C. Yanofsky, these PROCEEDINGS, 48, 173 (1962); Henning, U., and C. Yanofsky, ibid., 48, 183 (1962). 22 Pal6us, S., and H. Tuppy, Acta Chem. Scand., 13, 641 (1959). 23 Brown, H., F. Sanger, and R. Kitai, Biochem. J., 60, 556 (1955). 24 Harris, J. I., F. Sanger, and M. A. Naughton, Archiv. Biochem. and Biophys., 65, 427 (1956). 25 Matsubara, H., personal communication. " Nolan, C., and E. L. Smith, J. Biol. Chem., 237, 446, 453 (1962). 27 Lee, T. H., A. B. Lerner, and V. Buettner-Janusch, J. Biol, Chem., 236, 2970 (1961). Downloaded by guest on September 25, 2021 684 BIOCHEMIST'RY: SPEYER, LENGYEL, AND BASILIO PROC. N. A. S.

28 Katsoyannis, P. G., and V. duVigneaud, Arch. Biochem. and Biophysics, 78, 555 (1958). 29 1)ixon, J. S., and C. H. Li, Gen. and Comp. Endocrinol., 1, 161 (1961). 30 Acher, R., J. Chauvet, M. T. Lenci, F. Morel, and J. Maetz, Biochim. Biophys. Acta, 42, 379 (1960). 31 Chauvet, J., MI. T. Lenci, and R. Acher, Biochim. Biophys. Acta, 38, 671 (1960).

RIBOSOMAL LOCALIZATION OF STREPTOMYCIN SENSITIVITY* BY JOSEPH F. SPEYER, PETER LENGYEL, AND CARLOS BASILIot

DEPARTMENT OF BIOCHEMISTRY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE Communicated by Severo Ochoa, February 28, 1962 Erdds and Ullmann' reported that streptomycin inhibits the incorporation of labeled amino acids into protein in cell-free preparations of sensitive strains of My- cobacterium friburgensis but not in preparations of resistant strains. More recently Spotts and Stanier2 advanced the hypothesis that streptomycin sensitivity, resist- ance, or dependence, is the result of modifications in the structure of the ribosomes that affect their affinity for messenger-RNA. They propose that the structure of the ribosomes of sensitive cells endows them with a high affinity for streptomycin and that combination with this antibiotic interferes with the attachment of mes- senger-RNA with corresponding inhibition of protein synthesis. The ribosomes of resistant cells are supposed to have no affinity for streptomycin; consequently the drug does not affect their function. It had been found by Hancock3 that strepto- mycin sensitive, but not resistant, bacterial cells can bind small amounts of strep- tomycin irreversibly. We wish to report on experiments showing that inhibition of protein synthesis by streptomycin in cell-free preparations of sensitive strains of is due to interference of the drug with ribosomal function. Thus, streptomycin decreased the polyuridylic acid (poly U) dependent incorporation of C14-labeled phenyl- alanine into acid-insoluble products4 in a system containing (a) supernatant and ribosomes of sensitive, or (b) supernatant of resistant and ribosomes of sensitive E. coli. The drug had no effect with (a) supernatant and ribosomes of resistant, or (b) supernatant of sensitive and ribosomes of resistant cells. While these ex- periments point to the ribosomes as the site of streptomycin sensitivity, in agree- ment with the view of Spotts and Stanier, they throw no light on the actual mode of action of the drug. Preparations and Methods.-These were as previously described4 except that the preincubation of the ribosomes with supernatant, cold amino acids, and an generating system, was left out, and for the omission of cold amino acids (only phenylalanine-C14 being present) from the incubation mixture. Addition of ribosomes and streptomycin preceded that of the remaining components of the system. Streptomycin inhibition was less pronounced if the drug was added last. E. coli transfer RNA was added in saturating amounts. The incuba- tion was for 30 minutes at 37°. Poly U, sample 1 (sedimentation coefficient, 10. 3 S), was used. The amount of ribosomal protein, in a final volume of 0.25 ml, was approximately 0.78 mg in the case of streptomycin sensitive (E. coli W), and 0.61 mg in that of.streptomycin resistant cells. A culture of the latter cells (E. coli K12 F- 2341) was kindly provided by Dr. Alexander Tomasz, The Rockefeller Institute, New York. Wre are also indebted to Dr. David Perlman, The Squibb Downloaded by guest on September 25, 2021