Proc. Natl. Acad. Sci. USA Vol. 75, No. 1, pp. 275-279, January 1978 Biochemistry

Streptomycin causes misreading of natural messenger by interacting with ribosomes after initiation (protein synthesis/phenotypic suppression/temperature-sensitive aminoacyl-tRNA synthetase/phage R17 RNA translation/ aminoglycosides) PHANG-C. TAI, BRIAN J. WALLACE*, AND BERNARD D. DAVIS Bacterial Physiology Unit, Harvard Medical School, Boston, Massachusetts 02115 Contributed by Bernard D. Davis, November 10, 1977

ABSTRACT The induction of misreading by streptomycin conditions, misreading should promote replacement of the in vitro, previously observed with synthetic messengers, is now missing amino acid and hence should stimulate protein syn- demonstrated with natural (endogenous or viral) messenger by thesis. Such stimulation by Str was in fact observed with purified the use of extracts of temperature-sensitive mutants lacking polysomes (from either sensitive or resistant cells). Moreover, Glu-tRNA or Val-tRNA synthetase. With chain-elongating but noninitiating ribosomes (i.e., purified polysomes) deprived of with initiating sensitive ribosomes, stimulation was also observed an aminoacyl-tRNA, streptomycin and other aminoglycosides, but was restricted, like phenotypic suppression in sensitive cells, over a wide range of concentrations, stimulate incorporation. to a narrow range of low Str concentrations. It thus appears that With ribosomes initiating in the presence of streptomycin the paradoxical ability of Str to cause either misreading or stimulation is also observed but it is restricted, just like phe- cessation of protein synthesis in cells depends not on a different notypic suppression in cells, to very low streptomycin concen- response of the same ribosome to Str molecules at low or high trations which evidently allow some ribosomes to initiate and later encounter them in the course of chain elongation. The concentrations but rather on the relative frequency with which stimulation is accompanied by an increase in the size of the Str encounters an initiating or a chain-elongating ribosome. products; hence, it is evidently due to substitution of an incorrect In another contradiction, in earlier studies, Str at high con- aminoacyl-tRNA for a missing one. The test introduced here centrations caused phenotypic suppression in resistant (StrR) also has revealed a misreading effect of streptomycin on resis- cells (7, 8) but with resistant ribosomes in vitro it could not be tant ribosomes. In addition, significant intrinsic misreading was observed without streptomycin, indicating that under optimal shown to cause significant misreading of synthetic messenger conditions for in vitro protein synthesis an empty codon is fre- (3, 9, 10). With the more physiological system used in the quently read by an incorrect aminoacyl-tRNA. present work the expected misreading was easily demon- strated. Streptomycin (Str) has the remarkable property of producing two mutually exclusive effects with cells of sensitive (Strs) strains MATERIALS AND METHODS of Escherichia coli: high concentrations (>20,g/ml) com- Bacteria and Growth. RNase I-negative E. coli- strain pletely inhibit protein synthesis and rapidly lead to cell death MRE600 and a spontaneous StrR (200 ,ug/ml) derivative were (see ref. 1 for review), whereas low concentrations (e.g., 2 grown at 370 and used for the preparation of endogenous ,ug/ml) can cause phenotypic suppression of auxotrophic mu- polysomes free of initiation factors (IF) as described (6). Tem- tants (1, 2), which requires continued protein synthesis. The perature-sensitive E. coli mutants 10B6 (ThyA -, Val-ts) and results of efforts to study these phenomena in vitro have not 5F2 (ArgA-, Glu-ts) from Kaplan et al. (11) and NP29 (Val-ts) been entirely coherent. With synthetic messenger, continued from Eidlic and Neidhardt (12) were grown at 300 with aera- synthesis and misreading is observed over a broad range of tion in L broth supplemented with the salts of medium A (13). concentrations of Str (3) but with natural messenger, similar The temperature-sensitive cultures were incubated at 420 for concentrations block synthesis (4) and thus provide no apparent 30 min to inactivate the temperature-sensitive synthetase and opportunity for continued misreading. exhaust the corresponding aa-tRNAs. The cultures were then A possible resolution of this inconsistency was suggested by poured over ice, and the S100 and IF fractions were prepared the finding that Str encountering the ribosome before initiation from the cells (6) and stored at -76°. completely blocks translation (5), whereas with purified poly- Reagents and Buffers. Buffers, S30 extracts, S100 fractions, somes, whose ribosomes are already past initiation and cannot IF, and phage R17 RNA were prepared as described (6). An reinitiate (6), Str allows continued chain elongation (5) and thus equimolar mixture of 20 "4C-labeled amino acids, each at 10 has an opportunity for misreading. Moreover, under these Ci/mol, was prepared by adding the 6 required "4C-labeled conditions, translation is slowed, which implies some distortion amino acids to a mixture of 14 obtained from Amersham/ of the ribosome, and this distortion could well be associated with Searle. A mixture of 15 "4C-labeled amino acids (NEC-445, an increased frequency of errors. We have presented prelimi- composition of acid hydrolysate of alga) and all other ra- nary evidence in support of this hypothesis (1), based on stim- dioactive chemicals were from New England Nuclear. Str ulation of synthesis in the presence of only 15 amino acids. sulfate was from E. R. Squibb; neomycin, from Upjohn; kan- This paper presents the results of more detailed studies in amycin sulfate and paromomycin, from Bristol Laboratories; which deprival of an aminoacyl-tRNA (aa-tRNA) was and gentamicin sulfate, from Schering. Other reagents (reagent achieved more completely by using the extract of a mutant with grade) were obtained from commercial sources. a temperature-sensitive aa-tRNA synthetase. Under these Abbreviations: Str, streptomycin; Strs, streptomycin-sensitive; aa- The costs of publication of this article were defrayed in part by the tRNA, aminoacyl-tRNA; StrR, streptomycin-resistant; IF, initiation payment of page charges. This article must therefore be hereby marked factors. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate * Present address: School of Microbiology, The University of New this fact. South Wales, Kensington, New South Wales, Australia, 2033. 275 Downloaded by guest on September 25, 2021 276 Biochemistry: Tai et al. Proc. Natl. Acad. Sci USA 75 (1978)

A B c

fn 4- 12[ x E 8[

0

E. 0 4

C 0.2 gg/ml 20 jg/mI m ;, Av r To -AL - . v I --- f~ ~ ,- - -l- 0 0.1 1 10 100 10 15 20 10 15 20 10 15 20 Str, IAM Mg2+ mM FIG. 2. Effect of Str concentration on misreading on StrS (0) and FIG. 1. Effect of Str on peptide synthesis with polysomes de- StrR (0) polysomes. Reaction mixtures contained MRE600 StrS or prived of Glu-tRNA or Val-tRNA. Reaction mixtures (circles) con- StrR polysomes with 5F2 S100 and 9 mM Mg2+ as in Fig. 1A, with Str tained purified polysomes, 20 14C-labeled amino acids (each at 20 AM as specified. and 10 Ci/mol), S10T ctions from strains 5F2 (A), 10B6 (B), or MRE600 (C), and Str at 20 4/n~lwhere indicated (open symbol, with Str; solid symbol without Str). Incubation was for 20 min at 360. To to intrinsic misreading. To explore the effect of Str in a system demonstrate the absence of active Glu-tRNA synthetase or Val- with a sharper cutoff in the supply of specific aa-tRNAs, we tRNA synthetase, reaction mixtures (squares) with (closed squares) made use of E. coli mutants 5F2 and 10B6, whose tempera- or without (open squares) Str contained the 19 other unlabeled amino ture-sensitive aa-tRNA synthetase cannot acylate tRNAGlu or acids (at 50 MM each), together with S100 from 5F2 and 30 gM respectively, at a nonpermissive temperature (36°). [14C]glutamic acid (62 Ci/mol) (A), or S100 from 1OB6 and 30 AM tRNAval, [14Clvaline (85 Ci/mol) (B). (With the 5F2 preparation, no incorpo- As expected, at this temperature the use of the S100 fraction ration of [14C]glutamate was observed even at 290, which is a per- from 5F2 completely prevented incorporation of labeled glu- missive temperature in vivo; with the 1OB6 preparation, [14Clvaline tamate (in the presence of the 19 other unlabeled amino acids); incorporation was 45% as active as with the wild-type S100 at 290 and the same was true of labeled valine with the S100 from 10B6. optimal Mg2+, 9 mM.) Neither Str nor increased Mg2+ restored any incorporation (Fig. 1 A and B). In Vitro Protein Synthesis. Polypeptide synthesis was car- When all the amino acids were labeled,-there was appreciable ried out as described (6), at 360. The 1-ml reaction mixtures background incorporation with the 5F2 S100, highly dependent 60 NH4Cl, 20 mM contained 50 mM Tris.HCl (pH 7.6), mM on Mg2+ concentration (Fig. 1A, open circles). This synthesis KCl, 2 mM dithiothreitol, 1 mM ATP/Tris, 0.02 mM GTP, 5 evidently reflects the intrinsic incorporation of incorrect amino mM potassium phosphoenolpyruvate, 30 jig of pyruvate kinase, acids at Glu codons under these conditions in the absence of 0.1 ml of S100 fraction (about 0.8 mg of protein), 500 jig of Glu-tRNA. Addition of Str almost doubled the amino acid in- purified polysomes or washed ribosomes from MRE600 (as- corporation at 9 mM Mg2+ (the optimum for incorporation with suming 16.6 A260 units = 1 mg), 9 mM Mg(OAc)2 (unless oth- wild-type S100), and even with-the large intrinsic misreading erwise specified), and amino acids as specified. Phage R17 RNA observed at increased Mg2+ Str had a significant effect. Indeed, (500 jig/ml) and crude IF (200 jig/ml) were added as indicated, in the presence of Str the incorporation of 19 labeled amino in the amounts that yielded maximal activity. Samples were acids with the temperature-sensitive S100 was similar to the taken at intervals, incorporation was stopped by the addition incorporation of 20 amino acids with the wild-type S100 (Fig. of 2 ml of 5% trichloroacetic acid (6), and the precipitate was 1 A and C). assayed for radioactivity in toluene-based scintillation fluid with An S100 from temperature-sensitive mutant 10B6, which counter. a Nuclear-Chicago Isocap scintillation Background supported no incorporation of [14C]valine, yielded similar re- (200-300 cpm) without incubation was subtracted from all data sults except that the intrinsic synthesis without Str at low Mg2+ presented. was greater (Fig. 1B), and the pronounced inhibitory effect of Str on this high background resulted in net inhibition. However, RESULTS net stimulation was seen above 12 mM Mg2+. An S100 fraction We confirmed our earlier data (1) showing that, with purified prepared from another temperature-sensitive Val-tRNA syn- polysomes, Str stimulated incorporation in a protein-synthe- thetase mutant of E. coli, NP 29, gave identical results (data not sizing system with a mixture of 15 14C-labeled amino acids shown). (composition that of acid hydrolysate of alga). Str prolonged Streptomycin Concentration. As Fig. 2 shows, the extent of peptide synthesis and almost doubled the level achieved in 20 misreading on these Str-sensitive polysomes, like the previously min. Indeed, in the presence of Str at 20,jg/ml, incorporation observed slowing (6), reached a plateau at a low concentration was about 80% as great as in the complete system, both in initial of Str (0.2 jig/ml). [At high Mg2+ concentrations somewhat rate and in extent. It thus appears that, when ribosomes reach more Str was required for stimulation (data not shown).] Just a codon for a missing aa-tRNA, Str promotes the rapid substi- as in earlier studies with synthetic polynucleotides (14), the tution of another aa-tRNA, allowing the ribosomes to overcome extent of misreading did not vary up to 200 Aig of Str per ml a temporary block in chain elongation and continue peptide (Fig. 2; Table 1). synthesis, although at a reduced rate. Misreading Induced by Other Antibiotics. Because other Stimulation of Peptide Synthesis on Polysomes Limited aminoglycosides also promote misreading of synthetic poly- for a Specific aa-tRNA. The use of an incomplete amino acid nucleotides (3, 14, 15), several of these were compared with Str mixture has the disadvantage that the background incorpora- for their effect ohi the translation of natural messenger without tion is due in part to traces of the missing amino acids present Glu-tRNA synthetase. Gentamicin, neomycin C, paromomy- (and perhaps continuously released) in the extracts as well as cin, and kanamycin all stimulated incorporation (Table 1). Downloaded by guest on September 25, 2021 Biochemistry: Tai et al. Proc. Natl. Acad. Sci USA 75 (1978) 277

Table 1. Effect of type and concentration of antibiotics on peptide synthesis without Glu-tRNA Incorporation activity, % 1 lgIml 10 jg/ml 50 ug/ml 200 jsg/ml

Streptomycin 169 166 158 168 4-4 Gentamicin 192 243 324 184 x Neomycin C 201 148 111 Paromomycin 230 220 102 Kanamycin 203 169 2- E Reaction mixtures with purified polysomes, 20 14C-labeled amino acids, S100 from temperature-sensitive strain 5F2 (see Fig. 1A) and antibiotics as indicated were incubated for 20 min. Control without antibiotics (2360 cpm) was taken as 100% activity. 20 40 60 Time, min Unlike Str, however, the degree of stimulation varied with FIG. 3. Stimulation of peptide synthesis without Glu-tRNA on concentration, supporting the earlier suggestion (14) that these initiating ribosomes. Reaction mixtures (0.05 ml) contained StrS ri- compounds may act on more than one site on an elongating bosomes (washed with 1 M NH4Cl, then activated by exposure to 500 ribosome. for 2 min prior to use), phage R17 RNA, IF, S100 from 5F2 (lacking Misreading on StrR Ribosomes. With polysomes from a StrR Glu-tRNA), 0.1 MCi of a mixture of 15 14C-labeled amino acids, and 20 unlabeled amnino acids (each at 20 AM), in addition to other com- strain, Str stimulated amino acid incorporation in the absence ponents for peptide synthesis. Str (0) and gentamicin (3) were at 0.1 of Glu-tRNA nearly as much as with StrO polysomes (Fig. 2). and 50 gg/ml, respectively. 0, No antibiotic. Maximal stimulation required a much higher Str concentration (>20 ,gg/ml), as might be expected from the low affinity of StrR of small peptides. Addition of Str (at a low concentration) to the ribosomes for Str (16). The intrinsic misreading on StrR poly- synthesizing system greatly increased the average molecular somes was increased at increased Mg2+, much as with StrO size of the products. polysomes. Similar experiments were carried out with ribosomes ini- Misreading with Initiating Ribosomes. In the above ex- tiating on viral RNA, which offer the advantage of yielding periments with a noninitiating system, the stimulating effect defined products. In the control, with wild-type S100, sodium of Str was observed over a wide range of concentrations. Be- dodecyl sulfate/polyacrylamide gel analysis revealed three cause the higher concentrations completely block initiating peptides (Fig. 4A): a major peak of phage coat protein (fraction sensitive ribosomes (4, 6, 17), we have suggested (1) that the 55, molecular weight -14,000), a smaller peak (fraction 20) of prolonged misreading observed in growing cells might result RNA synthetase (18), and an unidentified peak at fraction 46. from intracellular Str concentrations low enough to fail to The system lacking Glu-tRNA (Fig. 4B) and without Str yielded contact (and block) many ribosomes during initiation but high much less labeled material, in which only one peak could be enough to encounter them later, during-chain elongation. detected, with a molecular weight close to that of coat protein To test this model in vitro, we examined the effect of low (fraction 55). (The analytical procedure would not have de- concentrations of Str on peptide synthesis by ribbsomes ini- tected small peptides.) The identity of the coat protein was tiating on viral messenger, in a system deprived of Glu-tRNA. confirmed by immunoprecipitation with antiserum (data not All 20 labeled amino acids were provided and the messenger shown). In the presence of a low concentration of1Str, the de- was in excess in order to prevent blockade of one ribosome by fective system produced about 50% more coat protein, with a another. In the control without antibiotic there was significant slightly broadened peak (Fig. 4C), and also a peak of higher spontaneous misreading of codons for glutamate (at 9 mM molecular weight (-30,000; fractions 31-45). (The origin of the Mg2+): incorporation of label was slow but it was still pro- latter peak is not clear; it might be due to "read through"-ie., ceeding at 60 min (Fig. 3). The rate of synthesis was stimulated misreading of termination triplets in the viral RNA.) Str also 1.5- to 2-fold by Str at 0.1 ug/ml and even more by gentamicin stimulated coat protein synthesis with StrR ribosomes. at 50 ,gg/ml [at which concentration protein synthesis in an These effects confirm the conclusion that the stimulation of initiating system on R17 RNA was not completely inhibited peptide synthesis by Str in a defective system depends on ac- (data not shown)]. As Table 2 shows, Str was stimulatory under celerated substitution of an incorrect amino acid for a missing these conditions over only a narrow concentration range, be- correct one. tween 0.02 and 0.4 ,gg/ml, with maximal effect at 0.1 ,g/ml; 1 ,ug/ml had negligible effect,,and 206,g/ml produced marked Table 2. Effect of Str concentration on initiating ribosomes inhibition (i.e., the stimulation of misreading was outweighed lacking Glu-tRNA by the blockade of initiation). Str, ,ug/ml Activity, % of control Products of Str-Stimulated Synthesis. The stimulation of translation of 0.02 116 natural messenger by Str, under the conditions 0.06 .135 described above, strongly suggests misreading of empty codons 0.1 (0.17 uM) 153 with consequent further chain elongation. However, the 0.4 142 stimulation could conceivably be due instead to accelerated 1 108 release of incomplete chains (and perhaps also to stimulation 2 104 of reinitiation). The two mechanisms should be distinguishable 10 88 by the length of the chains produced. 20 33 Initial experiments were performed, in the system without The reactions were carried out as in Fig. 3, except the incubation Glu-tRNA, with purified endogenous polysomes. The incor- was for 30 min and 0.1-ml samples were analyzed for radioactivity. poration product, analyzed on gel electrophoresis, was a smear The control without Str (2758 cpm) was taken as 100%. Downloaded by guest on September 25, 2021 278 Biochemistry: Tai et al. Proc. Natl. Acad. Sc USA 75 (1978)' with phage RNA (Fig. 4) and by molecular size distribution of the products obtained with endogenous messenger. The second paradox is that, in sensitive cells, low concen- trations of Str cause phenotypic suppression but high concen- trations stop protein synthesis altogether.Because Str binds in K vitro to a single site on the ribosome (16), over a wide range of E concentrations, a two-site explanation did not seem plausible. The double effect could be explained, however, if high con- centrations of Str blocked most initiating ribosomes whereas low concentrations often failed to block ribosomes at initiation and then encountered them (with resultant misreading) during 20 40 60 20 40 60 20 40 60 chain elongation. The present work confirms this explanation Fraction by demonstrating the predicted effect in vitro. Thus, with ri- FIG. 4. Size distribution of peptides formed on initiating ribo- bosomes initiating on viral RNA, in the system lacking Glu- somes with phage R17 RNA as messenger. Reaction mixtures were tRNA, peptide synthesis was stimulated by Str (Table 2) over as in Fig. 3 except for use of MRE600 S100 (A), temperature-sensitive a narrow range of low concentrations (0.1-0.4 ,g/ml, corre- 5F2 S100 (B), or 5F2 S100 plus Str at 0.1 ig/ml (C); 250-,Al samples sponding to a Str/ribosome ratio of 1:1 to 4:1). This model ex- were analyzed. After 60 min at 360, 2 ml of 6% trichloroacetic acid was plains an important early finding, with phage RNA in vitro, added and the precipitates were collected by centrifugation. To made before initiation in protein synthesis had been distin- eliminate adsorbed aa-tRNA and peptidyl-tRNA, the precipitates guished from were dissolved in 1 ml of 0.3 M NaOH, incubated at 36° for 10 min, chain elongation: Str stimulated, but only at low and mixed with 1.5 ml of 10% trichloroacetic acid. After centrifuga- concentrations, incorporation of a single added amino acid in tion, the precipitates were dissolved and subjected to sodium dodecyl an S80 deprived of asparagine by use of asparaginase (20). sulfate/phosphate/polyacrylamide gel electrophoresis as described The third paradox is that Str-induced misreading, as mea- (19), in 7.5% gels at 8 mA per tube for 6 hr. Slices (1 mm) were added sured by poly(U)-coded incorporation of isoleucine, was sub- to 0.3 ml of 95% Protosol and 8 ml of toluene-based scintillation fluid, stantial with StrS incubated at 370 overnight, and assayed for radioactivity. Molecular ribosomes but only negligible with StrR ribo- weight standards were run at the same time in a separate tube; the somes (3,9), even though the misreading effect of Str was dis- arrows in B are bovine serum albumin, 65,000; DNase I, 31,000; cy- covered on the basis of phenotypic suppression in StrR cells (7). tochrome, 12,570. However, in the present work, in a system lacking Glu-tRNA, Str caused almost as extensive misreading with Str as with StrO DISCUSSION polysomes, as measured by total translation of polysomes (Fig. The present paper clears up three paradoxes that have emerged 2) and by synthesis of coat protein coded for by phage RNA in studies on the inhibitory and the misreading actions of Str (data not shown). in vitro. The earlier failure to observe Str-stimulated misreading of First, over a wide range of concentrations, Str causes mis- poly(U) with StrR ribosomes appears to be due to an unex- reading in the translation of synthetic polynucleotide messen- plained peculiarity of the test chosen, isoleucine incorporation. gers (3, 14), although the same concentrations cause complete Using the same system, we have observed moderate Str-induced inhibition of the translation of viral RNA (4, 17). However, Str incorporation of leucine, but not of isoleucine or serine, by ri- causes only partial inhibition of the translation of natural bosomes from several StrR mutants (data not shown). Evidently, messenger (viral or endogenous) if it encounters ribosomes only the ability of various specific incorrect aa-tRNAs to misread after they have passed initiation and are engaged in chain a given codon can be altered by genetic changes in the structure elongation (5), or even if it is added to preformed fMet- of the ribosome. tRNA-ribosome-R17 RNA complexes (unpublished data). This paper has rounded out the evidence that the ability of Under these conditions, Str stimulates translation of purified Str to have both a misreading and an inhibitory effect at dif- polysomes supplied with only 15 amino acids, suggesting mis- ferent concentrations, in the cell and in vitro, depends on its reading (1). This paper provides more decisive evidence, ob- interaction with ribosomes in different stages of their cycle. tained under conditions such that the supply of aa-tRNA could Under some circumstances, Str can cause an increased rate be more sharply cut off by using an S100 with a temperature- of synthesis with poly(U) even in the presence of Phe-tRNA (3), sensitive aa-tRNA synthetase (for Glu-tRNA or for Val-tRNA). which has led to the suggestion (21) that an increased rate is the With these amino acid-deprived systems, Str over a concen- basis for the misreading effect of the antibiotic. However, our tration range from 0.2 to 200,ug/ml strongly stimulated peptide earlier experiments with purified polysomes, with 15 labeled synthesis on purified endogenous polysomes engaged in chain amino acids, led to the conclusion that Str slows rather than elongation without reinitiation (Figs. 1 and 2; Table 1). Indeed, accelerates synthesis on natural mRNA (5). In the present ex- in the presence of Str the incorporation of labeled amino acids periments, with all 20 amino acids labeled with the same spe- was nearly as rapid and as extensive in the incomplete as in the cific activity, total incorporation could be compared more di- complete system. rectly, and the results show that Str inhibits incorporation with Although the stimulation of synthesis in these incomplete wild-type S100. Thus, there is no doubt that, in the complete systems could be explained most readily by rapid misreading system under optimal conditions, total peptide elongation ac- at a codon for which the correct aminoacyl-tRNA was not tivity on natural mRNA is inhibited by Str, even though mis- available, it was also possible that on the purified polysomes the reading is stimulated. leading ribosome, blocked at an empty colon, might be holding The synthesis observed in our controls without Str inciden- up a sequence of ribosomes, and Str might be accelerating re- tally provides information on the rate and the extent of intrinsic lease of such a blocked ribosome from the polysome. The for- misreading of an empty codon in vitro. With an S100 with mer interpretation, in terms of misreading, was confirmed by temperature-sensitive Glu-tRNA or Val-tRNA synthetase there showing that Str stimulated the synthesis of longer chains, was no detectable incorporation of the corresponding amino identified by the increased formation of complete coat protein acids, but there was significant incorporation of 19 other amino Downloaded by guest on September 25, 2021 Biochemistry: Tai et al. Proc. Natl. Acad. Sci USA 75 (1978) 279

acids (Fig. 1 A and B). At the lowest Mg2a concentration 1. Davis, B. D., Tai, P.-C., & Wallace, B. J. (1974) in Ribosomes, eds. yielding virtually maximal translation of endogenous polysomes Nomura, M., Tissieres, A. Lengyel, P., (Cold Spring Harbor with wild-type S100 (9 mM; Fig. 1C), the glutamate-less syn- Laboratory, Cold Spring Harbor, NY), pp. 771-789. thesis was 14% of the normal whereas at higher Mg2+ concen- 2. Gorini, L. (1974) in Ribosomes, eds. Nomura, M., Tissieres, A. trations [as observed previously with synthetic polynucleotides & Lengyel, P. (Cold Spring Harbor Laboratory, Cold Spring 7)] the intrinsic misreading increased considerably, yielding Harbor, NY), pp. 791-803. (3, 3. Davies, J., Gilbert, W. & Gorini, L. (1964) Proc. Natl. Acad. Sci. over half as much incorporation as the wild-type S100 at 15 mM USA 51, 883-890. Mg2+. Moreover, with the same system initiating on viral RNA 4. Modolell, J. & Davis, B. D; (1968) Proc. NatI. Acad. Sci. USA 61, (at 9 mM Mg2+), synthesis was still proceeding slowly at 60 min 1279-1286. (Fig. 3); at that time it had reached about 7% of the total ob- 5. Wallace, B. J., Tai, P.-C., Herzog, E. L. & Davis, B. D. (1973) served (and completed within 30 min) with active Glu-tRNA Proc. NatI. Acad. Sci. USA 70, 1234-1237. synthetase (data not shown), and the labeled product had a peak 6. Tai, P.-C., Wallace, B. J., Herzog, E. L. & Davis, B. D. (1973) (Fig. 4B) with the molecular size and serological reactivity Biochemistry 12, 609-615. characteristic of the phage coat protein. These findings show 7. Gorini, L. & Kataja, E. (1964) Proc. NatI. Acad. Sci. USA 51, in of the ribosomes encountering 487-493. that, the'absence Glu-tRNA, 8. Gorini, L. & Kataja, E. (1965). Biochem. Biophys. Res. Commun. an empty codon are not rapidly released under the in vitro 18,656-663. conditions but often substitute, at a low rate, an incorrect aa- 9. van Knippenberg, P. H., van Ravenswaay Claasen, J. C., tRNA. In particular, the production of coat protein [which Grijm-Vox, M., Veldstra, H. & Bosch, L. (1965) Biochim. Biophys. contains five glutamate residues (22)] as a sharp peak rather Acta 95, 461-473. than as a broad range of molecular weights suggests that mis- 10. Anderson, W. F., Gorini, L. & Breckenridge, L. (1965) Proc. NatI. reading at an empty codon (instead of release) is quite frequent Acad. Sci. USA 54, 1076-1083. under the conditions used. (We have not specifically looked for 11. Kaplan, S., Atherly, A. G. & Varrett, A. (1973) Proc. Natl. Acad. smaller peptides that may have been produced at earlier times Sci. USA 70,689-692. and been degraded during the 60-min incubation.) 12. Eidlic, L. & Neidhardt, IF. C. (1965) J. Bacteriol. 89, 706-711. 13. Davis, B. D. & Mingioli, E. S. (1950) J. Bacteriol. 60, 17-28. Studies on misreading ultimately find their greatest interest 14. Davies, J. & Davis, B. D. (1968) J. Biol. Chem. 243, 3312- for the light that they may shed on the basis for the remarkable 3316. accuracy of normal translation in the cell, and the findings 15. Davies, J., Gorini, L. & Davis, B. D. (1965) Mol. Pharmacol. 1, presented in this paper indicate that such studies may be pur- 93-106. sued with natural messenger. It has been proposed that in- 16. Chang, F. N. & Flaks, J. G. (1972) Antimicrob. Agents Che- creased misreading depends on an effect on the equilibrium mother. 2, 294-307. of codon-anticodon binding (2, 23, 24) or, alternatively, on the 17. Luzzatto, L., Apirion, D. & Schlessinger, D. (1969) J. Bacteriol kinetics of chain elongation; the latter model was based on the 99,206-209. theoretical expectation that slowing of a discriminative step will 18. Capecchi, M. R. & Webster, R. E. (1975) in RNA Phages, ed. increase the effectiveness of the discrimination (25), possibly Zinder, N. D. (Cold Spring Harbor Laboratory, Cold Spring by affecting the dissociation rate (26) and nonspecific binding Harbor, NY), pp. 279-299. is 19. Shapiro, A. L., Vinuela, E. & Maizel, J. V., Jr. (1967) Biochem. energy (27). A third, more concrete basis for misreading Biophys. Res. Commun. 28,815-820. brought in with Hopfield's two-step model for recognition (28), 20. Schwartz, J. H. (1965) Proc. NatI. Acad. Sci. USA 53, 1133- which has been experimentally demonstrated by Thompson 1140. and Stone (29) in this laboratory. In this model, the initial, re- 21. Kurland, C. G. (1977) Annu. Rev. Biochem. 46, 173-200. versible binding of aa-tRNA, which frequently rejects incorrect 22. Weber, K. (1967) Biochemistry 6,3144-3154. species, is followed by a "kinetic proofreading" or "editing" 23. Gorini, L. (1971) Nature New Biol. 234,261-264. step in which energy from GTP hydrolysis is used in moving 24. Kurland, C. G., Rigler, R., Ehrenberg, M. & Blomberg, C. (1975) a correct aa-tRNA into an irreversibly bound state (see ref. 30). Proc. Natl. Acad. Sci. USA 72,4248-4251. This movement allows the ribosome a second chance to reject 25. Ninio, J. (1974) J. Mol. Biol. 84, 297-313. incorrect aa-tRNAs. It will be important to determine which 26. Ninio, J. (1975) Biochimie 57,587-595. 27. Blomberg, C. (1977) J. Theor. Biol. 66, 307-325. step is influenced by Str. 28. Hopfield, J. J. (1974) Proc. NatI. Acad. Sci. USA 71, 4135- We are grateful for the excellent technical assistance of Nancy 4139. Knight, Eve Arnold, and Su Jane Chen. This work was supported in 29. Thompson, R. C. & Stone, P. J. (1977) Proc. Nati. Acad. Sci. USA part by grants from the National Institutes of Health and the American 74, 198-202. Cancer Society (Massachusetts Division). 30. Lakes, J. A. (1977) Proc. Natl. Acad. Sci. USA 74, 1903-1907. Downloaded by guest on September 25, 2021