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Amber suppressors: Efficiency of chain propagation and suppressor specific amino acids
J. Mol. Biol. (1965) 14, 528-533
Amber Suppressors: Efficiency of Chain Propagation and Suppressor Specific Amino Acids S. KAPLAN,
A. O. W.
STRETTON AND S. BRENNER
Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, England (Received 20 September 1965) Previous experiments showed that sui inserts serine at the site of mutation in H36, an amber mutant of the head protein of bacteriophage T4D. The amino acids inserted by sutl and SUi'll are now shown to be glutamine and tyrosine, respectively. The efficiencies of chain propagation by these suppressors have been measured and are 63%, 51 % and 30% for sut, SUji and SUi'll'
1. Introduction
Amber mutants of the head protein of bacteriophage T4D produce N-terminal fragments of the polypeptide chain when grown in non-suppressing bacteria (Sarabhai, Stretton, Brenner & Bolle, 1964; Stretton & Brenner, 1965). In bacteria carrying the amber suppressor, su.~, the chain is propagated with the introduction of a serine residue at the site of the mutation (Stretton & Brenner, 1965). The same suppressor (Signer, Beckwith & Brenner, 1965; Garen, Garen & Wilhelm, 1965) also inserts serine in two amber mutants of the alkaline phosphatase gene (Weigert & Garen, 1965a) and in an amber mutant of the f2 bacteriophage (Notani, Engelhardt, Konigsberg & Zinder, 1965). Two other amber suppressors, SUl~ and SUl~I' are known which map on the bacterial chromosome in positions different from each other and from su i; SUI~ is linked to the gal genes and SU;rl is linked to the tryp genes (Signer et al., 1965; Garen et al., 1965; Brenner, unpublished results). From their patterns of suppression of bacteriophage mutants it appeared likely that the amino acids inserted by these suppressors would be different from each other and from serine. In this paper we show that su;r inserts glutamine and su;rr inserts tyrosine in agreement with the findings of Weigert, Lanka & Garen (1965). We also show that the three suppressors propagate the chain with different efficiencies.
2. Materials and Methods (a) Bacterial strains All strains are derivatives of Escherichia coli K12 Hfr H, and have been previously described (Brenner & Beckwith, 1965). CA154 is the su - strain, CA266, CA180 and CA265 contain sut, sutl and SUjil' respectively. (b) Bacteriophages T4D and the amber mutant H36 isolated by Dr R. H. Epstein (Stretton & Brenner, 1965) and H36Rl, a spontaneous revertant of amber H36 (Stretton & Brenner, unpublished results), were used. [l4C] Amino acids were purchased from the Radiochemical Centre, Amersham, Lysozyme, RNase, DNase, trypsin, chymotrypsin and leucine aminopeptidase were products of Worthington Biochemical Company, Freehold, N.J. 528
-
T4D
su
T4D
H36
su
sU I
+
H36
+
sUa
PhTI2
---~
+
[14 C] Phe
PLATE I. Autoradiographs of tryptic peptides of [14C]phenylalanine-labelled phage (ionophoresis at pH 6·4).
[facing p. 528
T4D su
PhTI2
H36
sut
H36 su~
• PhTI2Cb
• +
+
['"C]Phe
PLATE II. Autoradiograph of the [14C]phenylalanine-labelled chymotryptic digest of peptide PhTl2 (see Plate I) (ionophoresis at pH 6,4).
• T4D
H36
H36
su-
sui
su~
.-
PhTI2
.. P. A.l
•
PhTl2Cb
•
P. A.3
+ [ clPh e
PLATE III. Autoradiograph of the partial acid hydrolysate of peptide PhT12 Cb (see Plate II) (ionophoresis at pH 6,4).
..
Phe
+
..
PheGlu
•
AspPhe
w I SUn
..
+ [' · C] Phe
Edmon
P LATE IV. A u t oradiographs of the p eptid es PAl and PA:l (see Pla t e III ) after on e cycle of the Edman degradation (ionophor esis at pH 6·4).
-
TI 2
..
... I·e I Phe
Tyr
+ PLATE V. Autoradiograph of tryptic peptides of [14C]phenylalanine.labelled phage (ionophoresis at pH 6,4).
I
)
..
TI2
T4D
su
H36 su ~
Phe
+
'.
T4D
H36
RI
su
su~
su
Tyr
.
PLATE VI. Autoradiograph of peptide PhT1 2Tyr . The b and mar ke d T12Tyr fr om Plate V was re-run at pH 3·6.
)
T12T1 '
T4D
H36
su
sui Phe
H36
suilj Tyr
+
PLATE VI I. Autoradiograph of peptide P hTI 2. The b a n d marked T l2 from Plat e V was re -r un at p H 3·6. .
-
T40
T40
suitt
sut
-
PLATE VIII. Autoradiograph of digests of protein from cells infected with H36 and labelled with [14C]histidine. Ionophoresis of the digests was carried out at pH 6·4. The arrow marks the po sition of the transmitted peptide.
SUPPRESSOR SPECIFIC AMINO ACIDS
529
Phenylalanylglutamate and phenylalanylasparlate were purchased from the Cyclo Chemical Corporation, Los Angeles, Calif. Methods for the preparation, digestion and fractionation of radioactive protein labelled with [14C]amino acids have been described previously (Sarabhai et al., 1964; Stretton & Brenner, 1965). Radioactivity of peptides was measured by eluting the zone from paper with pyridine acetate buffer, pH 6·4; after drying on planchcts, the samples were counted in a Nuclear Chicago low background end-window counter to at least 2000 cts,
3. Results (a) Amino acids inserted by suiI and sujjr Tryptic digests of the fragment produced by amber H36 contain a hexapeptide, PhTll, comprising the first six amino acids of the wild-type tridecapeptide, PhT12, which has the sequence: Ala-Gly-Val- Phe-Asp- Phe-Gln-Asp.Pro-Ile-Asp- Ile-Arg. When amber H36 is grown on su 1, a peptide corresponding to PhT12 can be isolated, with a serine residue in place of the glutamine at position 7 (Stretton & Brenner, unpublished results). Since the amino acid sequence of PhT12 is known, it is possible to devise experimental procedures to determine the amino acid at this position when the mutant is grown on strains carrying other amber suppressors. The amino acid inserted by snjj The following experiments allowed us to conclude that the amino acid inserted by this suppressor must be asparagine, glutamine or phenylalanine. (i) Amber H36 was grown on CA180 and the protein synthesized was labelled with [14C]phenylalanine. Tryptic digests contained a peptide with the same electrophoretic mobility as PhTl2 (Plate I). This result showed that the charged amino acids, lysine, arginine, histidine, glutamic and aspartic acids, and also tyrosine (see below) were not inserted by SUI~' (ii) In separate experiments, the suppressed product was labelled with [14C]trypto. phan, [14C]serine, [14C]threonine and [14C]leucine, amino acids which are not present in wild-type PhT12. The suppressed PhT12 was not labelled, eliminating these amino acids. A similar experiment with 35S04 eliminated methionine and cysteine. (iii) The suppressed protein was then labelled in separate experiments with [14 e } alanine, [14C]glycine and [14C]valine. The peptide corresponding to PhT12 was purified and digested with chymotrypsin which splits PhT12 into two fragments, Cl , the neutral N-terminal tetrapeptide, Ala-Gly-Val-Phe, and C2, the acidic C·terminal nonapeptide (Stretton & Brenner, 1965). C2 was not labelled in these experiments, which eliminated alanine, glycine and valine. (iv) The suppressed peptide was labelled with [14C]isoleucine and, after purification, digested with leucine aminopeptidase which releases the first seven amino acids from PhT12. No free isoleucine was found. In another experiment, the peptide was labelled with [14C]proline and digested with leucine aminopeptidase. Since proline interrupts the sequential action of the enzyme, if proline had been inserted, the extent of degradation would be less and the resulting proline-containing peptide would be larger. The peptide had the same electrophoretic mobility which eliminated proline. The amino acid inserted by su!r was shown to be glutamine by the following experimental procedure. The proteins synthesized by T4D on the su - strain, and by amber H36 on the su 1 and sUJ~ strains, were labelled with [14C]phenylalanine, digested 34
530
S. KAPLAN, A. O. W. STRETTON AND S. BRENNER
with trypsin and fractionated by ionophoresis at pH 6·4 (Plate I). The peptide zones corresponding to PhTI2 were removed, purified by a second ionophoresis at pH 3·6 and digested with chymotrypsin. Ionophoresis at pH 6·4 separated peptide CI from C2, the acidic C-terminal nonapeptide (Plate II). The acidic peptides were eluted and partially hydrolysed with 6 N-HCI for two days at 37°C. This treatment also completely deamidates glutamine and asparagine. Plate III shows that the partial hydrolysate of C2 from H36 grown on SUI~ is identical with that of the wild type and that both differ from the partial hydrolysate of C2 from H36 grown on su;. This is direct evidence that an amide is inserted by SUI~' The peptide marked PAl in Plate III has the sequence Asp-Phe and yields free phenylalanine after one cycle of the Edman degradation (Plate IV). From the mobility of this peptide it is possible to calculate the mobility of the peptide Asp-Phe-Glu expected in the wild type. PA3 corresponds to this peptide. The peptide in H36 grown on SUI~ could be either Asp-Phe-Glu or Asp-Phe-Asp. Both PA3 peptides were eluted and one cycle of the Edman degradation performed. Plate IV shows that the wild type and the suppressed amber mutant gave identical products which migrate more slowly than Asp-Phe. This suggests that the structure in both cases is Phe-GIu, a result which was directly confirmed by measuring the electrophoretic mobilities of synthetic Phe-Glu and Phe-Asp (Fig. I). We conclude that the amino acid inserted by su~ is glutamine.
0
_
Phe Glu Phe Asp
+ FIG. 1. A tracing of the positions of phenylalanine (Phe), phenylalanylglutamate (Phe-Glu.) and phenylalanylaspartate (Phe-Asp.) after ionophoresis at pH 6·4. The compounds were located by staining with ninhydrin.
The amino acid inserted by SUI~I The determination of the amino acid inserted by this suppressor was simplified by the initial observation that the [14C]phenylalanyl-labelled peptide corresponding to PhTI2 in the suppressed protein had an altered -electrophoretic mobility, moving slightly behind the wild-type PhTI2. A similar result had been previously observed
SUPPRESSOR SPECIFIC AMINO ACIDS
531 Tyr
in a spontaneous revertant of H36, H36RI, which contains a peptide, PhT12 , having tyrosine at position 7 (Stretton & Brenner, unpublished results). This result suggested that tyrosine is inserted by SUI~I' and the following experiment confirms this conclusion. The proteins synthesized by H36 in S11I~I' and by T4D and H36RI in the su - strain, were separatcly labelled with [14C]phenylalanine and [14C]tyrosinc. Tryptic digests were fractionated by ionophoresis at pH 6·4 (Plate V). The zones corresponding to PhTI2 of the wild type and to PhTI2 T y r of H36RI were removed and subjected to ionophoresis at pH 3·6. Plate VI shows clearly that PhTI2 is present only in the wild type and is absent both in the revertant and in H36 grown in SUI~I' Plate VII shows that a peptide with the same electrophoretic mobility as PhTI2 T y r is present in the suppressed product, and that this peptide, in addition, contains tyrosine. We conclude that SUI~I inserts tyrosine. (b) Efficiency of the amber suppressors Stretton & Brenner (1965) reported that when amber H36 is grown in s'U,i chain termination is not completely suppressed and both the fragment and the complete chain are made. The fraction of chains propagated is a direct measure of the efficiency of the suppressor. This can be determined by comparing the amounts of peptides distal (C-terminal) to the amber site (transmitted peptides) with the amounts of peptides proximal (N-terminal) to the site (standard peptides) for any given suppressing strain. The results of one experiment are presented in Table I. In this experiment, the proteins synthesized by amber H36 in the su.- and the three su.+ strains and wild-type protein were labelled with [14C]histidine, denatured, digested in parallel with chymotrypsin and subjected to ionophoresis at pH 6·4. The result is shown in Plate VIII; the arrow indicates the transmitted peptide which is present in the wild type and in H36 grown on the three suppressing strains, but is absent in the mutant grown in the su - . Zones corresponding to the transmitted peptide, and to three standard peptides known to be proximal to the H36 site, were eluted and counted. The first column of Table 1 gives the average of the three standard peptides (s) and the second column, TABLE
1
Transmission of H36 in suppressor strains
Phage
Strain
Ctsjmin average three standard peptides
Ctsjmin transmitted peptide
t/8
215 216 500 873 756 647 952
0,379 0'371 0·735 0,975 1·112 1·505 1·568
t]« corrected'[
%
transmission
8
H36
CAI54
H36 H36 H36 T4D
CA180 8U,~ CA265 CA266 BUt CAI54 8U-
8U-
su,;,
568t 583t 680 895 680 430t 588t
0 0·360 0,600 0·737 1·171
0 30 51 63 100
t The duplicate values were obtained from completely independent experiments. t 1/8 is corrected by subtracting 0,375, the average of the two values for H36 grown on
8U-.
532
S. KAPLAN, A. O. W. STRETTON AND S. BRENNER
the transmitted peptide (t). The ratio tle is shown in the third column, and, in the fourth column, this ratio has been corrected by subtracting the t/s found for H36 grown on the su. -. In this case, no band corresponding to the transmitted peptide can be seen, and the radioactivity found in the paper is background contamination. From the corrected tls ratios the percentage transmission is calculated by setting the wild type at 100%. Although we cannot reliably estimate the limits of accuracy of these measurements, in seven independent experiments, values ranging between 58 and 67% have been obtained for H36 grown on strains carrying su t. In one experiment, using amber B278 and sut, an efficiency of 65% was determined. The value of 30% for S'UI~ hal' been determined in three separate experiments, but the value of 51 % for SUI~I represents the only determination. TABLE
2
Summary of information on amber suppressors Suppressor
...
Map position linked to
Efficiency of chain propagation
Amino acid inserted
SUI
his l .a
63%4
serine 5 •6 •7
8Ul~
gaP
30%4 51%4
glutamine 4.s tyrosinev-"
... SUllI
t ryp l .3 References:
1. Garen et al. (1965) Signer et al. (1965)
2. 3. 4. 5. 6. 7. 8.
Brenner, unpublished results Present paper Notani et al. (1965) Stretton & Brenner (1965) Weigert & Garen (1965a) Weigert et al. (1965)
4. Discussion Table 2 contains a summary of the map positions, efficiencies of propagation and the amino acids inserted by the three amber suppressors. Our results on the amino acids inserted by SUI~ and SUI~I are in exact agreement with those of Weigert et al. (1965). We may safely conclude that it is likely that each amber suppressor will insert the same amino acid in all amber mutants. The interesting feature of these results is that the amino acids inserted by the three suppressors are coded by triplets which are all connected to the amber triplet by single base changes. Evidence has been presented that the amber triplet is DAG and the suppressor amino acids are amongst those which are found as revertants of the amber triplet (Weigert & Garen, 1965b; Brenner, Stretton & Kaplan, 1965). These results are consistent with the hypothesis that the different amber suppressors are due to single base mutations in the anti-codons of sRNA's of serine, glutamine and tyrosine which normally code for DCG, CAG and UAD or UAC, respectively. Recently, Cappechi & Gussin (1965) have shown that suppression in su t might involve an altered serine sRNA. Naturally, this hypothesis implies that normal reading of the amino acid triplets must be preserved. Either there are multiple sRNA's reading the same
SUPPRESSOR SPECIFIC AMINO ACIDS
533
triplet, or there are multiple ways of reading the same triplet. Crick (personal communication) has pointed out that there may be one sRNA which does not distinguish between A and G in the third position of the triplet and another which reads G specifically, and for triplets with U and C in the third position there could be a specific reading of the U in addition to an ambiguous reading. The G or U specific sRNA's may then be altered, without lethal consequence. If the hypothesis is correct, and if the provision of an alternative reading for triplets is universal, we might expect to find a maximum of seven different amber suppressors; the remaining four would insert leucine, tryptophan, lysine and glutamic acid. Finally, according to this hypothesis, the efficiency of suppression is a measure ofthe relative activities ofthe three amino acyl sRNA's when matched against the competing sRNA which we have postulated to be involved in chain termination (Brenner et al., 1965). If the activities measured are directly related to amounts of sRNA, we could conclude that the relative amounts of the four sRNA's are: chain terminating, 1; serine, 2; glutamine, 0·5; tyrosine, 1. This work was carried out while one of us (S.K.) was a Postdoctoral Fellow, Division of General Medical Sciences National Institutes of Health. REFERENCES Brenner, S. & Beckwith, J. R. (1965). J. Mol. Riol. 13, 629. Brenner, S., Stretton, A. O. W. & Kaplan, S. (1965). Nature, 206, 994. Cappechi, M. & Gussin, G. (1965). Science, 149, 417. Garen, A., Garen, S. & Wilhelm, R. C. (1965). J. Mol. Riol. 14, 167. Notani, G. W., Engelhardt, D. L., Konigsberg, W. & Zinder, N. (1965). J. Mol. Riol. 12, 439. Sarabhai, A. S., Stretton, A. O. W., Brenner, S. & Bolle, A. (1964). Nature, 201, 13. Stretton, A. O. W. & Brenner, S. (1965). J. Mol. Riol. 12, 456. Weigert, M. G. & Garen, A. (1965a). J. Mol. Riol. 12, 448. Weigert, M. G. & Garen, A. (1965b). Nature, 206, 992. Weigert, M. G., Lanka, E. & Garen, A. (1965). J. Mol. Riol. 14, 522.
Amber Suppressors: Efficiency of Chain Propagation and Suppressor Specific Amino Acids S. KAPLAN,
A. O. W.
STRETTON AND S. BRENNER
Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, England (Received 20 September 1965) Previous experiments showed that sui inserts serine at the site of mutation in H36, an amber mutant of the head protein of bacteriophage T4D. The amino acids inserted by sutl and SUi'll are now shown to be glutamine and tyrosine, respectively. The efficiencies of chain propagation by these suppressors have been measured and are 63%, 51 % and 30% for sut, SUji and SUi'll'
1. Introduction
Amber mutants of the head protein of bacteriophage T4D produce N-terminal fragments of the polypeptide chain when grown in non-suppressing bacteria (Sarabhai, Stretton, Brenner & Bolle, 1964; Stretton & Brenner, 1965). In bacteria carrying the amber suppressor, su.~, the chain is propagated with the introduction of a serine residue at the site of the mutation (Stretton & Brenner, 1965). The same suppressor (Signer, Beckwith & Brenner, 1965; Garen, Garen & Wilhelm, 1965) also inserts serine in two amber mutants of the alkaline phosphatase gene (Weigert & Garen, 1965a) and in an amber mutant of the f2 bacteriophage (Notani, Engelhardt, Konigsberg & Zinder, 1965). Two other amber suppressors, SUl~ and SUl~I' are known which map on the bacterial chromosome in positions different from each other and from su i; SUI~ is linked to the gal genes and SU;rl is linked to the tryp genes (Signer et al., 1965; Garen et al., 1965; Brenner, unpublished results). From their patterns of suppression of bacteriophage mutants it appeared likely that the amino acids inserted by these suppressors would be different from each other and from serine. In this paper we show that su;r inserts glutamine and su;rr inserts tyrosine in agreement with the findings of Weigert, Lanka & Garen (1965). We also show that the three suppressors propagate the chain with different efficiencies.
2. Materials and Methods (a) Bacterial strains All strains are derivatives of Escherichia coli K12 Hfr H, and have been previously described (Brenner & Beckwith, 1965). CA154 is the su - strain, CA266, CA180 and CA265 contain sut, sutl and SUjil' respectively. (b) Bacteriophages T4D and the amber mutant H36 isolated by Dr R. H. Epstein (Stretton & Brenner, 1965) and H36Rl, a spontaneous revertant of amber H36 (Stretton & Brenner, unpublished results), were used. [l4C] Amino acids were purchased from the Radiochemical Centre, Amersham, Lysozyme, RNase, DNase, trypsin, chymotrypsin and leucine aminopeptidase were products of Worthington Biochemical Company, Freehold, N.J. 528
-
T4D
su
T4D
H36
su
sU I
+
H36
+
sUa
PhTI2
---~
+
[14 C] Phe
PLATE I. Autoradiographs of tryptic peptides of [14C]phenylalanine-labelled phage (ionophoresis at pH 6·4).
[facing p. 528
T4D su
PhTI2
H36
sut
H36 su~
• PhTI2Cb
• +
+
['"C]Phe
PLATE II. Autoradiograph of the [14C]phenylalanine-labelled chymotryptic digest of peptide PhTl2 (see Plate I) (ionophoresis at pH 6,4).
• T4D
H36
H36
su-
sui
su~
.-
PhTI2
.. P. A.l
•
PhTl2Cb
•
P. A.3
+ [ clPh e
PLATE III. Autoradiograph of the partial acid hydrolysate of peptide PhT12 Cb (see Plate II) (ionophoresis at pH 6,4).
..
Phe
+
..
PheGlu
•
AspPhe
w I SUn
..
+ [' · C] Phe
Edmon
P LATE IV. A u t oradiographs of the p eptid es PAl and PA:l (see Pla t e III ) after on e cycle of the Edman degradation (ionophor esis at pH 6·4).
-
TI 2
..
... I·e I Phe
Tyr
+ PLATE V. Autoradiograph of tryptic peptides of [14C]phenylalanine.labelled phage (ionophoresis at pH 6,4).
I
)
..
TI2
T4D
su
H36 su ~
Phe
+
'.
T4D
H36
RI
su
su~
su
Tyr
.
PLATE VI. Autoradiograph of peptide PhT1 2Tyr . The b and mar ke d T12Tyr fr om Plate V was re-run at pH 3·6.
)
T12T1 '
T4D
H36
su
sui Phe
H36
suilj Tyr
+
PLATE VI I. Autoradiograph of peptide P hTI 2. The b a n d marked T l2 from Plat e V was re -r un at p H 3·6. .
-
T40
T40
suitt
sut
-
PLATE VIII. Autoradiograph of digests of protein from cells infected with H36 and labelled with [14C]histidine. Ionophoresis of the digests was carried out at pH 6·4. The arrow marks the po sition of the transmitted peptide.
SUPPRESSOR SPECIFIC AMINO ACIDS
529
Phenylalanylglutamate and phenylalanylasparlate were purchased from the Cyclo Chemical Corporation, Los Angeles, Calif. Methods for the preparation, digestion and fractionation of radioactive protein labelled with [14C]amino acids have been described previously (Sarabhai et al., 1964; Stretton & Brenner, 1965). Radioactivity of peptides was measured by eluting the zone from paper with pyridine acetate buffer, pH 6·4; after drying on planchcts, the samples were counted in a Nuclear Chicago low background end-window counter to at least 2000 cts,
3. Results (a) Amino acids inserted by suiI and sujjr Tryptic digests of the fragment produced by amber H36 contain a hexapeptide, PhTll, comprising the first six amino acids of the wild-type tridecapeptide, PhT12, which has the sequence: Ala-Gly-Val- Phe-Asp- Phe-Gln-Asp.Pro-Ile-Asp- Ile-Arg. When amber H36 is grown on su 1, a peptide corresponding to PhT12 can be isolated, with a serine residue in place of the glutamine at position 7 (Stretton & Brenner, unpublished results). Since the amino acid sequence of PhT12 is known, it is possible to devise experimental procedures to determine the amino acid at this position when the mutant is grown on strains carrying other amber suppressors. The amino acid inserted by snjj The following experiments allowed us to conclude that the amino acid inserted by this suppressor must be asparagine, glutamine or phenylalanine. (i) Amber H36 was grown on CA180 and the protein synthesized was labelled with [14C]phenylalanine. Tryptic digests contained a peptide with the same electrophoretic mobility as PhTl2 (Plate I). This result showed that the charged amino acids, lysine, arginine, histidine, glutamic and aspartic acids, and also tyrosine (see below) were not inserted by SUI~' (ii) In separate experiments, the suppressed product was labelled with [14C]trypto. phan, [14C]serine, [14C]threonine and [14C]leucine, amino acids which are not present in wild-type PhT12. The suppressed PhT12 was not labelled, eliminating these amino acids. A similar experiment with 35S04 eliminated methionine and cysteine. (iii) The suppressed protein was then labelled in separate experiments with [14 e } alanine, [14C]glycine and [14C]valine. The peptide corresponding to PhT12 was purified and digested with chymotrypsin which splits PhT12 into two fragments, Cl , the neutral N-terminal tetrapeptide, Ala-Gly-Val-Phe, and C2, the acidic C·terminal nonapeptide (Stretton & Brenner, 1965). C2 was not labelled in these experiments, which eliminated alanine, glycine and valine. (iv) The suppressed peptide was labelled with [14C]isoleucine and, after purification, digested with leucine aminopeptidase which releases the first seven amino acids from PhT12. No free isoleucine was found. In another experiment, the peptide was labelled with [14C]proline and digested with leucine aminopeptidase. Since proline interrupts the sequential action of the enzyme, if proline had been inserted, the extent of degradation would be less and the resulting proline-containing peptide would be larger. The peptide had the same electrophoretic mobility which eliminated proline. The amino acid inserted by su!r was shown to be glutamine by the following experimental procedure. The proteins synthesized by T4D on the su - strain, and by amber H36 on the su 1 and sUJ~ strains, were labelled with [14C]phenylalanine, digested 34
530
S. KAPLAN, A. O. W. STRETTON AND S. BRENNER
with trypsin and fractionated by ionophoresis at pH 6·4 (Plate I). The peptide zones corresponding to PhTI2 were removed, purified by a second ionophoresis at pH 3·6 and digested with chymotrypsin. Ionophoresis at pH 6·4 separated peptide CI from C2, the acidic C-terminal nonapeptide (Plate II). The acidic peptides were eluted and partially hydrolysed with 6 N-HCI for two days at 37°C. This treatment also completely deamidates glutamine and asparagine. Plate III shows that the partial hydrolysate of C2 from H36 grown on SUI~ is identical with that of the wild type and that both differ from the partial hydrolysate of C2 from H36 grown on su;. This is direct evidence that an amide is inserted by SUI~' The peptide marked PAl in Plate III has the sequence Asp-Phe and yields free phenylalanine after one cycle of the Edman degradation (Plate IV). From the mobility of this peptide it is possible to calculate the mobility of the peptide Asp-Phe-Glu expected in the wild type. PA3 corresponds to this peptide. The peptide in H36 grown on SUI~ could be either Asp-Phe-Glu or Asp-Phe-Asp. Both PA3 peptides were eluted and one cycle of the Edman degradation performed. Plate IV shows that the wild type and the suppressed amber mutant gave identical products which migrate more slowly than Asp-Phe. This suggests that the structure in both cases is Phe-GIu, a result which was directly confirmed by measuring the electrophoretic mobilities of synthetic Phe-Glu and Phe-Asp (Fig. I). We conclude that the amino acid inserted by su~ is glutamine.
0
_
Phe Glu Phe Asp
+ FIG. 1. A tracing of the positions of phenylalanine (Phe), phenylalanylglutamate (Phe-Glu.) and phenylalanylaspartate (Phe-Asp.) after ionophoresis at pH 6·4. The compounds were located by staining with ninhydrin.
The amino acid inserted by SUI~I The determination of the amino acid inserted by this suppressor was simplified by the initial observation that the [14C]phenylalanyl-labelled peptide corresponding to PhTI2 in the suppressed protein had an altered -electrophoretic mobility, moving slightly behind the wild-type PhTI2. A similar result had been previously observed
SUPPRESSOR SPECIFIC AMINO ACIDS
531 Tyr
in a spontaneous revertant of H36, H36RI, which contains a peptide, PhT12 , having tyrosine at position 7 (Stretton & Brenner, unpublished results). This result suggested that tyrosine is inserted by SUI~I' and the following experiment confirms this conclusion. The proteins synthesized by H36 in S11I~I' and by T4D and H36RI in the su - strain, were separatcly labelled with [14C]phenylalanine and [14C]tyrosinc. Tryptic digests were fractionated by ionophoresis at pH 6·4 (Plate V). The zones corresponding to PhTI2 of the wild type and to PhTI2 T y r of H36RI were removed and subjected to ionophoresis at pH 3·6. Plate VI shows clearly that PhTI2 is present only in the wild type and is absent both in the revertant and in H36 grown in SUI~I' Plate VII shows that a peptide with the same electrophoretic mobility as PhTI2 T y r is present in the suppressed product, and that this peptide, in addition, contains tyrosine. We conclude that SUI~I inserts tyrosine. (b) Efficiency of the amber suppressors Stretton & Brenner (1965) reported that when amber H36 is grown in s'U,i chain termination is not completely suppressed and both the fragment and the complete chain are made. The fraction of chains propagated is a direct measure of the efficiency of the suppressor. This can be determined by comparing the amounts of peptides distal (C-terminal) to the amber site (transmitted peptides) with the amounts of peptides proximal (N-terminal) to the site (standard peptides) for any given suppressing strain. The results of one experiment are presented in Table I. In this experiment, the proteins synthesized by amber H36 in the su.- and the three su.+ strains and wild-type protein were labelled with [14C]histidine, denatured, digested in parallel with chymotrypsin and subjected to ionophoresis at pH 6·4. The result is shown in Plate VIII; the arrow indicates the transmitted peptide which is present in the wild type and in H36 grown on the three suppressing strains, but is absent in the mutant grown in the su - . Zones corresponding to the transmitted peptide, and to three standard peptides known to be proximal to the H36 site, were eluted and counted. The first column of Table 1 gives the average of the three standard peptides (s) and the second column, TABLE
1
Transmission of H36 in suppressor strains
Phage
Strain
Ctsjmin average three standard peptides
Ctsjmin transmitted peptide
t/8
215 216 500 873 756 647 952
0,379 0'371 0·735 0,975 1·112 1·505 1·568
t]« corrected'[
%
transmission
8
H36
CAI54
H36 H36 H36 T4D
CA180 8U,~ CA265 CA266 BUt CAI54 8U-
8U-
su,;,
568t 583t 680 895 680 430t 588t
0 0·360 0,600 0·737 1·171
0 30 51 63 100
t The duplicate values were obtained from completely independent experiments. t 1/8 is corrected by subtracting 0,375, the average of the two values for H36 grown on
8U-.
532
S. KAPLAN, A. O. W. STRETTON AND S. BRENNER
the transmitted peptide (t). The ratio tle is shown in the third column, and, in the fourth column, this ratio has been corrected by subtracting the t/s found for H36 grown on the su. -. In this case, no band corresponding to the transmitted peptide can be seen, and the radioactivity found in the paper is background contamination. From the corrected tls ratios the percentage transmission is calculated by setting the wild type at 100%. Although we cannot reliably estimate the limits of accuracy of these measurements, in seven independent experiments, values ranging between 58 and 67% have been obtained for H36 grown on strains carrying su t. In one experiment, using amber B278 and sut, an efficiency of 65% was determined. The value of 30% for S'UI~ hal' been determined in three separate experiments, but the value of 51 % for SUI~I represents the only determination. TABLE
2
Summary of information on amber suppressors Suppressor
...
Map position linked to
Efficiency of chain propagation
Amino acid inserted
SUI
his l .a
63%4
serine 5 •6 •7
8Ul~
gaP
30%4 51%4
glutamine 4.s tyrosinev-"
... SUllI
t ryp l .3 References:
1. Garen et al. (1965) Signer et al. (1965)
2. 3. 4. 5. 6. 7. 8.
Brenner, unpublished results Present paper Notani et al. (1965) Stretton & Brenner (1965) Weigert & Garen (1965a) Weigert et al. (1965)
4. Discussion Table 2 contains a summary of the map positions, efficiencies of propagation and the amino acids inserted by the three amber suppressors. Our results on the amino acids inserted by SUI~ and SUI~I are in exact agreement with those of Weigert et al. (1965). We may safely conclude that it is likely that each amber suppressor will insert the same amino acid in all amber mutants. The interesting feature of these results is that the amino acids inserted by the three suppressors are coded by triplets which are all connected to the amber triplet by single base changes. Evidence has been presented that the amber triplet is DAG and the suppressor amino acids are amongst those which are found as revertants of the amber triplet (Weigert & Garen, 1965b; Brenner, Stretton & Kaplan, 1965). These results are consistent with the hypothesis that the different amber suppressors are due to single base mutations in the anti-codons of sRNA's of serine, glutamine and tyrosine which normally code for DCG, CAG and UAD or UAC, respectively. Recently, Cappechi & Gussin (1965) have shown that suppression in su t might involve an altered serine sRNA. Naturally, this hypothesis implies that normal reading of the amino acid triplets must be preserved. Either there are multiple sRNA's reading the same
SUPPRESSOR SPECIFIC AMINO ACIDS
533
triplet, or there are multiple ways of reading the same triplet. Crick (personal communication) has pointed out that there may be one sRNA which does not distinguish between A and G in the third position of the triplet and another which reads G specifically, and for triplets with U and C in the third position there could be a specific reading of the U in addition to an ambiguous reading. The G or U specific sRNA's may then be altered, without lethal consequence. If the hypothesis is correct, and if the provision of an alternative reading for triplets is universal, we might expect to find a maximum of seven different amber suppressors; the remaining four would insert leucine, tryptophan, lysine and glutamic acid. Finally, according to this hypothesis, the efficiency of suppression is a measure ofthe relative activities ofthe three amino acyl sRNA's when matched against the competing sRNA which we have postulated to be involved in chain termination (Brenner et al., 1965). If the activities measured are directly related to amounts of sRNA, we could conclude that the relative amounts of the four sRNA's are: chain terminating, 1; serine, 2; glutamine, 0·5; tyrosine, 1. This work was carried out while one of us (S.K.) was a Postdoctoral Fellow, Division of General Medical Sciences National Institutes of Health. REFERENCES Brenner, S. & Beckwith, J. R. (1965). J. Mol. Riol. 13, 629. Brenner, S., Stretton, A. O. W. & Kaplan, S. (1965). Nature, 206, 994. Cappechi, M. & Gussin, G. (1965). Science, 149, 417. Garen, A., Garen, S. & Wilhelm, R. C. (1965). J. Mol. Riol. 14, 167. Notani, G. W., Engelhardt, D. L., Konigsberg, W. & Zinder, N. (1965). J. Mol. Riol. 12, 439. Sarabhai, A. S., Stretton, A. O. W., Brenner, S. & Bolle, A. (1964). Nature, 201, 13. Stretton, A. O. W. & Brenner, S. (1965). J. Mol. Riol. 12, 456. Weigert, M. G. & Garen, A. (1965a). J. Mol. Riol. 12, 448. Weigert, M. G. & Garen, A. (1965b). Nature, 206, 992. Weigert, M. G., Lanka, E. & Garen, A. (1965). J. Mol. Riol. 14, 522.