Amber suppression and activating enzymes

J. Mol. Biol. (1966) 16, 556--561

Amber Suppression and Activating Enzymes The polypeptide chain termination associated with amber mutations can be partially suppressed in certain strains of bacteria (Sarabhai, Stretton, Brenner & Bolle, 1964; Stretton & Brenner, 1965; Kaplan, Stretton & Brenner, 1965). In these suppressing strains (su +), I believe the amber codon (UAG) to be translated ambiguously: either as an amino acid specific for the suppressor; or as a command for polypeptide chain termination (Brenner, Stretton & Kaplan, 1965; Brenner & Stretton, 1965). Chain termination always occurs in non-suppressing (su -) strains. An attractive hypothesis, as yet unproved, is that there normally exists a specific transfer RNA to recognize the amber codon. A possible mechanism for the ambiguous translation of UAG might then involve the alteration of an amino acyl-tRNA t synthetase, so that in the su + strain the suppressor-specific amino acid can be loaded on the hypothesized amber tRNA, as well as the usual substrate tRNA(s). Amino acyl-amber tRNA would insert the amino acid in the growing polypeptide, which would be completed normally except for an amino acid substitution at the site of the amber mutation. For three strong amber suppressors (sui, sU;r, suiir ), the experiments described below have failed to detect any significant loading of the suppressor-specific amino acid on su + tRNA by su + activating enzymes that could not be reversed by eu.: enzymes. The results suggest that the mechanism for amber suppression proposed above is not correct, and that amber suppression is due instead to a mutant form of su + tRNA or ribosomes. Pairs of Escherichia coli strains the genetic complements of which are identical except for the suppressor under study were kindly supplied by Dr S. Brenner: SUr, which inserts serine, strains D21 (su -) and D21-4 (su SUll' which inserts glutamine, strains CA150 (su-) and CA161 (su;r); sUrII> which inserts tyrosine, strains CA244 (su-) and CA265 (surir) (Stretton & Brenner, 1965; Kaplan et al., 1965). S-100, used as the source of amino acyl-tRNA synthetases, was prepared from cells grown on 3XD medium (Fraser & Jerrel, 1953) as described by Sanger, Bretscher & Hocquard (1964) except that NH 4Cl was used instead ofKCl in the buffers. Commercial baker's yeast S-100 was a gift from Dr J. D. Smith. Transfer RNA was prepared from cells harvested and lysed according to Brubaker & McCorquodale (1963); for further purification the method described by Zubay (1962) was followed, including stripping amino acids from the tRNA at high pH. In the case of suii and surtr tRNA, the iso-propanol differential precipitation was omitted and a passage over DEAE-cellulose (Holley et al., 1961) was substituted. L-[14C]serine (87,4 mojm-mole), L-[14C]tyrosine (164 mc/m-rnole) and L-[14C]glutamine (32,2 mo/m-mole) were obtained from the Radiochemical Centre, Amersham. [35S]methionine (268 mojm-mole) was a gift from Dr K. Marcker. Figure 1 illustrates the time course of loading amino acids on tRNA. To minimize competition for the amber tRNA between the labelled amino acid and any other end group, non-radioactive amino acids were not added to the loading reactions.

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FIG. 1. Time course of loadiog the suppressor-specific amioo acid on BU+ tRNA. Reaction mixtures were iocubated at 30°0 and contained, per mI.: 50 p.moles potassium cacodylate (pH 7,0), 5 p.moles MgOl2 , 100 p.moles NH40l, 4·4 p.moles phosphoenolpyruvate, 1·2 p.moles ATP, 0·05 p.mole OTP, 6 p.moles ,B-mercaptoethanol, 20 p.g pyruvate kioase, 0·10 ml. of the appropriate 8-100 and either 1'8 mg BUt tRNA and 0·035 p.mole [140]serioe (-e-e-), or 2·6 mg 8U~r tRNA and 0·038 p.mole [140]glutamine (-0-0-), or 2·3 mg BUrt tRNA and 0·035p.mole [14C]tyrosioe (-~-~-).

The amino acyl-tRNA was isolated by adding 0·11 volume of 2 M-potassium acetate (pH 5'0) and extracting with 80% phenol (+20% 0·2 M-potassium acetate, pH 5,0). Three cycles of ethanol precipitation (two volumes of 95% ethanol) and resuspension in 0·2 M-potassium acetate (pH 5,0) were performed, the final resuspension in 0·02 Mpotassium acetate (pH 5'0) being followed by dialysis overnight against 1000 volumes of 0·002 M-potassium acetate (pH 5,0). Unloading (see legend to Fig. 3) was carried out at 30°C until the levels of cold acidinsoluble radioactive material could no longer be measured accurately or until the rate of unloading became nearly equal to that in the absence of enzymes. Samples were removed at various times and either precipitated on 1 cm X 3 em strips of Whatman 3MM filter paper by 5% trichloroacetic acid plus 3% Casamino acids (Bollum, 1959) at O°C and counted in a liquid-scintillation spectrometer (Nuclear Chicago), or precipitated in 5% trichloroacetic acid plus 3% Casamino acids, plated on Millipore filters and counted in a low-background counter (Nuclear Chicago). The rationale of the experiment is to load the suppressor-specific amino acid, in the presence of the 8U+ enzymes and an ATP-generating system, on 8U+ tRNA-not only the normal tRNA's specific for the amino acid, but also the hypothesized tRNA that recognizes the amber codon. Mter purifying the amino acyl-tRNA, the loading reaction is run in reverse in the presence of AMP, inorganic pyrophosphate and either 8U+ or 8U - enzymes. The 8U - enzymes should not be able to unload the amino acyl-amber tRNA. It is possible to demonstrate that the activating enzyme specificity controls unloading by making use of the fact that the methionyl synthetase derived from yeast can load methionine on only part of the E. coli tRNA loadable by the E. coli synthetase (Berg, Bergmann, Ofengand & Dieckmann, 1961). Figure 2 shows the time course of unloading E. coli [35S]methionyl-tRNA, loaded with E. coli enzymes, in the

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FIG. 2. Time course of unloading [ 3 58]methionyl.E. coli tRNA. The reaction mixture is described in Fig. 3, except that the yeast reactions contained 0·25 ml. of yeast 8-100. E. coli (strain CA161) tRNA was loaded with [ 3 58]methionine for 60 min at 30°C in the presence of E. coli 8-100. The purified amino acyl-tRNA was unloaded at 30°C in the presence of either E. coli 8-100 ( - . - . - ) or yeast 8-100 (-0-0-). The curves have been corrected for the exponential loss of acid. precipitable radioactive material in the absence of enzymes. At 60 min, 0·10 ml. E. coli 8-100 was added, per mI., to two of the yeast-unloading reactions. The approximately 8% of the label which cannot be unloaded by E. coli 8-100 is due to the formation of N-formyl methionyltRNA during loading (Marcker, 1965).

presence of E. coli enzymes or enzymes derived from yeast. There is a fraction of about 25% of the E. coli loadable [35SJmethionyl-tRNA that cannot be unloaded by yeast. In addition, Herve & Ohapeville (1963) have shown that the alanyl-tRNA produced by Raney nickel reduction of cysteinyl-tRNA cannot be unloaded by either the alanyl or the cysteinyl synthetase. Since one cannot depend on isolating enzymes of the same activity from various sources, the rate of discharge is not significant. Only therelative amounts of unload. able radioactive material at the end of the reaction indicate differences in enzyme specificity. In Fig. 3 are shown the time courses of unloading suppressor-specific amino acyl-su + tRNA's in the presence of either su + or su - enzymes. The curves have been corrected for loss of acid-insoluble radioactive material in the absence of enzymes (measured half-lives at pH 7'0, 30°0: for seryl.sui tRNA, 260 minutes; for glutaminyl.suir tRNA, 200 minutes; for tyrosyl-surir tRNA, 230 minutes; formethionyl-E. coli (strain OA161) tRNA, 150 minutes). There is no significant measurable difference in the amount of amino acid unloaded by su + and su - enzymes. At the end of the incubation,

LETTE R S TO THE EDITOR

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F IG. 3. Time course of unloading suppressor -sp ecific amino acyl -se + tRNA in the presence of au + or au - enzymes. E ach au + t R NA was loaded for 20 min 88 described in Fig. ·1 with the suppressor -specific a mino ac id in the presen ce of au + S-100 , and purified as described in t he t ext. Unloading r eactions were in cubated at 30°0 and contained , per ml.: 100 i£tnoles potassium cacodyl at e (pH 7'0), 10 JLmo les MgC12, 50 JLmoles N~ Cl , 5 JLmoles AMP, 5 I'moles potassium pyrophosphate, 6 i£moles ,B-mercaptoethan ol, 0·01 JLmole of the [12C]supp ressor-specific amino acid, 0·10 ILmole of the appropriate S·IOO and either 420 ILg [14C]seryl-au; tRNA (mlLmoles serine/mg tRNA ) (-e-e-), or 1400 ILg [140]glutaminyl- autI tRNA (0'2 mlLmole/mg tRNA) (- 0 - 0-), or 600 ILg [ U C]tyrosyl-suI~r t RNA (0'7 mjzm ole t yrosine/mg t RNA) (- A - A-). The roman numerals d esign ating t he curves have a subscri pt + or - t o indicat e whe t he r su+ or su S- 100 was present during the unloading. The curves have been corrected for t he exponential loss of ac id -inso lub le radioact ive material in t he abse nce of en zymes.

differences are less than 2% of the t otal glut aminyl-su ;I tRNA and less than t % of the t ot al seryl-su 1 t R NA and t yrosyl-8ur11 tRNA. Unloadin g was followed until less than 6% of the original label was acid-insoluble in t he case of glutaminyl-szsj] tRNA, less than 2% in the cases of seryl- su1 tRNA and tyrosyl-sur1r tRNA. The quantit at ive conclusions drawn from these results assume t hat all the radioa cti ve mat erial on the amino acyl-tRNA is in the load ed amino acid. Because glutamine 36

560

J. R. MENNINGER

can be so easily deaminated to glutamic acid, glutaminyl.sutI tRNA loaded for 20 minutes was analysed by stripping the amino acid from the tRNA in 0·04 M-NH40H at 37°0 for 45 minutes, and subjecting it to ionophoresis at pH 3·6 and then again at pH 8·9. The mobility of the bulk of the radioactive material removed was the same as for glutamine and quite different from glutamic acid, Or from the predicted mobility of iso.glutamine. The amount of glutamic acid contamination was measured by cutting out the appropriate areas of the ionogram and counting in a scintillation spectrometer; it was found to be 12%. Failure to detect unloadable amino acyl-amber tRNA may be attributable to there being too little of it, either because amber tRNA or its synthetases are lost or inactivated during extraction, or because insufficient opportunity for loading the suppressor-specific amino acid was provided. If the amber tRNA or its synthetase were especially labile, it should be impossible to demonstrate suppression in vitro; but Oapecchi & Gussin (1965) have already reported such suppression by the cell-free system of an amber mutation in the f2 bacteriophage coat protein gene. Furthermore, Takanami & Yan (1965) and Menninger (unpublished results) have shown that the 8U~ cell-free system incorporates a disproportionately large amount of serine, relative to other amino acids, when stimulated by a poly UAI or poly UAG messenger, whereas the su.- system does not. If an amber tRNA were bound specifically to ribosomes, it might be present in complete cell-free systems yet not be detectable in unloading experiments. Rosset, Monier & Julien (1964) have reported a low molecular weight ribosome-bound RNA, but in the presence of 8U; enzymes no measurable amount of serine (less than 1% of that loadable on "5 s" RNA-free tRNA) was loaded on this "5 s" RNA. The question of whether ample opportunity was given for loading the hypothesized amber tRNA can be answered in part by carrying out loading for 60 instead of for 20 minutes. Unloading this material gave the same results as those reported in Fig. 3. Figure 1 shows that in the case of glutamine loading, a significant amount of radioactive material was made acid-insoluble between 20 and 60 minutes of incubation. Seventy per cent of the increase could be prevented, however, by adding either an equimolar amount of [1 20]glutamic acid or equimolar amounts of nineteen [ 1 20]amino acids to the loading reaction. It therefore seems likely that most of the additional incorporated radioactivity is due to a slow transformation of glutamine into glutamic acid. Including nineteen [1 20]amino acids in the serine or tyrosine loading reactions did not affect the fraction of acid-insoluble radioactive material added between 20 and 60 minutes. Since in the complete cell-free assays referred to above, ambertRNA would only have to be present in catalytic amounts, I cannot rule out too small quantities (less than 8% of glutaminyl-t.RNe., less than 2% of seryl or tyrosyl-tRNA) as a reason for the negative results of the unloading experiments. Subject to this reservation, I feel that the unloading experiments show that, for the three suppressors studied, strong amber suppression cannot be due to an increase in specificity of an amino acyl.tRNA synthetase. Thus it is probable that either the 8U + tRNA or ribosomes are responsible, a conclusion which is supported by the demonstration by Capecchi & Gussin (1965) that an altered 8U; seryl-tRNA is responsible for suppression of an amber mutant in the f2 bacteriophage coat protein gene.

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I am a Research Fellow of the Helen Hay Whitney Foundation. I should like to thank Drs S. Brenner, F. H. C. Crick and other colleagues for many stimulating discussions;

L ETTER S TO THE E D I T O R

56 1

, V. A. Why brow and Mrs B . Mit chell for technical assistance; and Dr R. Monier for a sample of E . coli " 5 s" RNA and " 5 s " RNA-free tRN A . Medical R esearch Council Laborat ory of Molecul ar Biology Hills Road, Cambridge, England

J OHN

R.

MEN NIN GER

Received 24 November 1965

REFERENCE S B erg, P., Bergmann, F. H., Ofengand, E. J . & Di eckmann, M. (1961). J. B ioI. Ohem, 236, 1726 . B ollum, F. J. (1959). J. tu«: Chem . 234, 2733. Brenner , S. & Stre t to n, A . O. W. (1965). J. Mol. tu«. 13, 944. Brenner, S., Strett on , A . O. W. & K aplan, S. (1965). Nature, 206 , 994. Brubaker, L. H. & McCorquodale, D. J. (1963). B iochim. biophys. A cta, 76, 48. Capecchi, M. R & Gussin, G. N. (1965) . Science, 149, 417. Fraser, D. & Jerrel, E . A. (1953). J. B ioI. Chern. 205 , 291. H erve, G. & Chapeville, F . (1963). B iochim. biophys. A cta, 76, 493. H oll ey , R . W ., Ap gar, J ., D oct or, B. P ., F arrow, J., Marini, M. A. & Merrill, S. H. (1961). J. tu«. Ohern, 236, 200 . Kaplan, S., St retto n, A. O. W . & Brenner , S. (1965) . J . M ol. tu«. 14, 528. Marcker, K. (1965). J. Mol. tu«. 14, 63. Rosset , R, Monier, R & Julien , J. (1964). Bull. Soc. Chim . B ioI. 46, 87. Sanger, F ., Bret sch er , M. S. & H ocquard, E. J. (1964) . J. Mo l. tu«. 8, 38. Sarabhai, A. S., Stretto n, A . O. W ., Brenner, S. & B oll e, A. (1964). N ature , 201 , 13. St retton, A . O. W . & Brenner, S. (1965). J. Mol. B ioI. 12, 456. Takanami, M. & Yan, Y. (1965). Proc. Nat. Acad. sa; Wash. 54, 1450. Zubay, G. (1962). J. M ol. BioI. 4, 347.