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Amino acid substitutions resulting from suppression of nonsense mutations
J. Mol. Riol. (1965) 14, 522-527
Amino Acid Substitutions resulting from Suppression of Nonsense Mutations II. Glutamine Insertion by the 5u-2 Gene; Tyrosine Insertion by the 5u-3 Gene MARTIN G. WEIGERT, ERICH LANKA AND.ALAN GAREN
Department of Molecular Biology and Biophysics Yale University, New Haven, Oonn., U.S.A. (Received 13 July 1965) The NI class of phosphatase nonsense mutations is suppressible by either the Su-L, Su-2 or Su-3 suppressor genes of E8cherichia coli. It was previously shown that suppression by the Su-L gene results in the incorporation of a serine residue into the phosphatase molecule at the position specified by a nonsense codon. The present experiments demonstrate that the amino acid incorporated is glutamine when Su-2 is the suppressor gene, and is tyrosine when Su-3 is the suppressor gene. Thus, the same nonsense codon can be translated in three ways, as serine, glutamine or tyrosine, depending on which of the three suppressor genes is responsible for suppression.
1. Introduction There are several different suppressor genes in Escherichia coli that act on nonsense mutations (Garen, Garen & Wilhelm, 1965; Gallucci & Garen, 1966; Signer, Beckwith & Brenner, 1965). Suppression of a nonsense mutation may produce an alteration in the protein which is specified by the gene containing the nonsense mutation. It was reported earlier that suppression of a phosphatase nonsense mutation of the Nl class by the Su-1 suppressor gene resulted in the formation of phosphatase protein eontaining serine in place of an amino acid residue normally present at one position in the molecule (Weigert & Garen, 1965a). The amino acid residue that was replaced could vary, depending on which phosphatase mutation was suppressed, but in all cases the new amino acid inserted into the phosphatase molecule was serine. Thus, in the presence of the Su-1 gene the codon generated by an Nl nonsense mutation codes for serine. Similar results have been obtained for suppression of a nonsense mutation in an RNA phage by the Su-1 gene (Notani, Engelhardt, Konigsberg & Zinder, 1965) and for suppression of nonsense mutations in phage T4 by an analogous suppressor gene (Stretton & Brenner, 1965). The present report identifies the amino acids inserted into phosphatase protein as a result of the action of two other suppressor genes, Su·2 andSu-3 (Garen et al., 1965). The Su-2 and Su-3 genes suppress the same class of Nl phosphatase nonsense mutations as the Su-1 gene, but a different amino acid is inserted by each of the genes. 622
SUPPRESSION OF NONSENSE MUTATIONS
523
2.MareriahandMeilio~
The bacterial suppressor strains and the phosphatase nonsense mutants are described elsewhere (Garen et al., 1965). All of the chemical procedures used in the present experiments are described in an earlier report (Weigert & Garen, 1965a).
3. Results Phosphatase proteins isolated from suppressed strains of three phosphatase nonsense mutants were analyzed for amino acid substitutions. It was previously shown for one of the mutants, G5, that suppression by the Su-L suppressor gene caused the substitution of a serine residue for a glutamine residue normally present in the phosphatase tryptic peptide 9 (Weigert & Garen, 1965a; see Table 1). Analyses of peptide 9 from phosphatase protein obtained from G5 mutants suppressed by the Su-2 and Su-3 suppressor genes are shown in Table 1. Glutamine is the amino acid inserted into peptide 9 by the Su-2 gene, and tyrosine by the Su-3 gene. Suppression of another phosphatase nonsense mutant, H12, by the Suo] suppressor gene results in the replacement of the tryptophan residue of phosphatase tryptic peptide 1 by a serine residue (Weigert & Garen, 1965a; see Table 2). The effect of suppression of H12 by the Su-3 suppressor gene, as shown in Table 2, is the substitution of tyrosine for the tryptophan in peptide 1. It has not been possible to determine the effect of the Su-2 gene on H12 because the low efficiency of suppression of H12 by this gene (Garen et al., 1965) makes purification of phosphatase protein prohibitively difficult. The mutant S4:5 has been used to determine the amino acid inserted by the action of the suppressor gene present in strain WI (which corresponds to the widely used suppressor strain C600). By genetic criteria, the suppressor gene in WI appears to be identical to the Su-2 gene (Garen, unpublished results). It has been shown that a glutamine residue in tryptic peptide 21 of the standard protein is replaced by serine when S45 is suppressed by the Suo] gene (Weigert & Garen, 1965a; see Table 3). The analyses in Table 3 indicate that suppression of S45 by strain WI results in the incorporation of a glutamine residue into peptide 21. Thus, the amino acid analysis supports the genetic evidence indicating that the WI and Su-2 suppressor genes are identical.
4. Discussion The RNA triplet UAG has been identified as a nonsense codon involved both with the Nl class ofphosphatase nonsense mutations (Weigert & Garen, 1965b) and with the corresponding class of amber nonsense mutations in phage T4: (Brenner, Stretton & Kaplan, 1965). The present experiments demonstrate that this triplet exhibits a remarkable range of coding specificity. In anSu - strain it behaves as a nonsense codon, whereas in an Su + strain it may code either for serine, glutamine or tyrosine, depending on which suppressor gene, Su-I, Su-2 or Su-3, is responsible for suppression (see also the accompanying report by Kaplan, Stretton & Brenner, 1965). It has recently been shown that the carrier of suppressor activity in an Suo] strain is a serine transfer-RNA molecule (Capecchi & Gussin, 1965; Wilhelm, Engelhardt, Webster, Garen & Zinder, manuscript in preparation). Thus, the coding specificity of a nonsense codon in the Su-L strain depends on the formation of a
TABLE
1
Amino acid substitutions in peptide 9 resulting from supp ression of the mutation G5 Amino ac id composition of peptide 9 Strain
Standard P+ Suppressed G5 Suppressed G5 Suppressed G5
Substitution
Suppressor gene
Su.] Su·2 Su-3
Lys
Trpt
Thr
GIy
Ala
Phe
Glu:j:
Tyr§
Serll
1·00 1·00 1·00 1·00
+ + + +
2·00 2·30 1·94 2·36
2·04 2·20
2·82 3·04 2·74 3·01
0·87 0·87 0·95 0·96
2·81 2·00
-
0·91
1·96 2·00
2·90 1·83
-
-
0·85
-
glutamine ~ serine glutam ine ~ glutamine glutam ine --+ tyrosine
The amino add analyses were performed as described in an earlier report (Weigert & Ga ren, 1965a). All results a re normalized to a lysine value of 1·00. t Tryptophan was det erm ined qualitatively by treating the fingerprint paper with a specific stain for tryptophan. :j: The amino acid analys is does not distinguish between glut a m ine and glutamic acid. T he conclusion t hat glutamine is the residue in t he standard prot ein which is affected by suppression is b ased on the elect rop hore t ic behavior of the proteins. Since the proteins obt ained from the Su-L andSu-2 suppressor strains, which h av e serine and tyrosine sub st it utions, respectively, are not elect r op horet ically different from the standard protein, it is concluded that the original amin o acid in the standard protein which is r eplaced by serine and tyrosine is glutamine rather than glutamic acid. § There was some destruction of tyrosine during hydrolysis of t he peptide, which in these analyses amounted to a bout 10%. The value in the Table has not been corrected for this loss. II There was some destruction of serine du ring hydrolysis of the peptide, amounting t o 9 % . The value in tho Table has not been corrected for this loss.
TABLE
2
Amino acid substitutions in peptide 1 resulting from suppression of the mutation H12 Amino acid composition of peptide 1 Strain
Substitution
Suppressor gene Lys'[
Trp
1·00
+
Standard P+ Suppressed H12
sa.:
1·00
Gly
Leu
0·98
0·90
0·86
1·19
Amino acid composition of peptides 1 Standard P+ Suppressed H12
Su·3
Ser
1·05
+2
Lys'[
Trp
Giy
Leu
Ala
2·00
+
1·96
2·96
2·04
2·08
2·84
2·04
2·00
tryptophan --+ serine
Tyrt 0·56
tryptophan --+ tyrosine
Peptides 1 and 2 are sufficiently separated in the fingerprint of the protein obtained from the Su-L strain to permit a separate analysis of peptide 1. However, in the protein obtained from the Su·3 strain, peptides 1 and 2 overlap and have been analyzed together (for details, see Weigert & Garen, 1965b). t Peptides 1 and 2 each contain a single lysine residue. In the analyses of peptide 1 alone, the results are normalized to a value of 1·00 for lysine. In the combined analyses of peptides 1 and 2, the results are normalized to a value of 2·00 for lysine. t The tyrosine loss during hydrolysis of the peptide in these analyses was about 23%. The value in the Table has not been corrected for this loss.
TABLE
3
Amino acid substitutions in peptide 21 resulting from suppression. of the mutation 845 Amino acid composition of peptide 21 Strain
Standard P+ Suppressed S45 Suppressed S45
Suppressor gene
Su-1
WI
Substitution Arg
Asp
1·00 1·00 1·00
2·04 2·13 2·17
Thr
3·29 3·07 3·62
Gly
2·51 2·37 1·89
The conditions for the analyses are as described in the preceding Tables.
Ala
7·05 6·6 1 7·15
Val
2·97 3·03 3·10
Leu
3·23 2·72 3·36
H is
0·99 0·75 1·07
GIu
2·14 1·25 1·97
Ser
1·85 2·80 2·05
glutamine -->- serine glutamine -->- glu t amine
SUPPRESSION OF NONSENSE MUTATIONS
527
transfer-RNA which can accept serine and insert it in the position specified by the nonsense codon. Further studies of the biochemical effects of mutations affecting suppression should provide useful information about the genetic control of coding. REFERENCES Brenner, S., Stretton, A. O. W. & Kaplan, S. (1965). Nature, 206, 994. Capecchi, M. & Gussin, G. (1965). Science, 149, 417. Gallucci, E. & Garen, A. (1966). J. Mol. Biol. in the press. Garen, A., Garen, S. & Wilhelm, R. C. (1965). J. Mol. Biol. 14, 167. Kaplan, S., Stretton, A. O. W. & Brenner, S. (1965). J. Mol. Biol. 14, 528. Notani, G. W., Engelhardt, D. L., Konigsberg, W. & Zinder, N. (1965). J. Mol. Biol.12, 439. Signer, E. R., Beckwith, J. R. & Brenner, S. (1965). J. Mol. Biol. 14, 153. Stretton, A. O. W. & Brenner, S. (1965). J. Mol. Biol. 12, 456. Weigert, M. & Garen, A. (1965a). J. Mol. Biol. 12, 448. Weigert, M. & Garen, A. (1965b). Nature, 206,992.
Amino Acid Substitutions resulting from Suppression of Nonsense Mutations II. Glutamine Insertion by the 5u-2 Gene; Tyrosine Insertion by the 5u-3 Gene MARTIN G. WEIGERT, ERICH LANKA AND.ALAN GAREN
Department of Molecular Biology and Biophysics Yale University, New Haven, Oonn., U.S.A. (Received 13 July 1965) The NI class of phosphatase nonsense mutations is suppressible by either the Su-L, Su-2 or Su-3 suppressor genes of E8cherichia coli. It was previously shown that suppression by the Su-L gene results in the incorporation of a serine residue into the phosphatase molecule at the position specified by a nonsense codon. The present experiments demonstrate that the amino acid incorporated is glutamine when Su-2 is the suppressor gene, and is tyrosine when Su-3 is the suppressor gene. Thus, the same nonsense codon can be translated in three ways, as serine, glutamine or tyrosine, depending on which of the three suppressor genes is responsible for suppression.
1. Introduction There are several different suppressor genes in Escherichia coli that act on nonsense mutations (Garen, Garen & Wilhelm, 1965; Gallucci & Garen, 1966; Signer, Beckwith & Brenner, 1965). Suppression of a nonsense mutation may produce an alteration in the protein which is specified by the gene containing the nonsense mutation. It was reported earlier that suppression of a phosphatase nonsense mutation of the Nl class by the Su-1 suppressor gene resulted in the formation of phosphatase protein eontaining serine in place of an amino acid residue normally present at one position in the molecule (Weigert & Garen, 1965a). The amino acid residue that was replaced could vary, depending on which phosphatase mutation was suppressed, but in all cases the new amino acid inserted into the phosphatase molecule was serine. Thus, in the presence of the Su-1 gene the codon generated by an Nl nonsense mutation codes for serine. Similar results have been obtained for suppression of a nonsense mutation in an RNA phage by the Su-1 gene (Notani, Engelhardt, Konigsberg & Zinder, 1965) and for suppression of nonsense mutations in phage T4 by an analogous suppressor gene (Stretton & Brenner, 1965). The present report identifies the amino acids inserted into phosphatase protein as a result of the action of two other suppressor genes, Su·2 andSu-3 (Garen et al., 1965). The Su-2 and Su-3 genes suppress the same class of Nl phosphatase nonsense mutations as the Su-1 gene, but a different amino acid is inserted by each of the genes. 622
SUPPRESSION OF NONSENSE MUTATIONS
523
2.MareriahandMeilio~
The bacterial suppressor strains and the phosphatase nonsense mutants are described elsewhere (Garen et al., 1965). All of the chemical procedures used in the present experiments are described in an earlier report (Weigert & Garen, 1965a).
3. Results Phosphatase proteins isolated from suppressed strains of three phosphatase nonsense mutants were analyzed for amino acid substitutions. It was previously shown for one of the mutants, G5, that suppression by the Su-L suppressor gene caused the substitution of a serine residue for a glutamine residue normally present in the phosphatase tryptic peptide 9 (Weigert & Garen, 1965a; see Table 1). Analyses of peptide 9 from phosphatase protein obtained from G5 mutants suppressed by the Su-2 and Su-3 suppressor genes are shown in Table 1. Glutamine is the amino acid inserted into peptide 9 by the Su-2 gene, and tyrosine by the Su-3 gene. Suppression of another phosphatase nonsense mutant, H12, by the Suo] suppressor gene results in the replacement of the tryptophan residue of phosphatase tryptic peptide 1 by a serine residue (Weigert & Garen, 1965a; see Table 2). The effect of suppression of H12 by the Su-3 suppressor gene, as shown in Table 2, is the substitution of tyrosine for the tryptophan in peptide 1. It has not been possible to determine the effect of the Su-2 gene on H12 because the low efficiency of suppression of H12 by this gene (Garen et al., 1965) makes purification of phosphatase protein prohibitively difficult. The mutant S4:5 has been used to determine the amino acid inserted by the action of the suppressor gene present in strain WI (which corresponds to the widely used suppressor strain C600). By genetic criteria, the suppressor gene in WI appears to be identical to the Su-2 gene (Garen, unpublished results). It has been shown that a glutamine residue in tryptic peptide 21 of the standard protein is replaced by serine when S45 is suppressed by the Suo] gene (Weigert & Garen, 1965a; see Table 3). The analyses in Table 3 indicate that suppression of S45 by strain WI results in the incorporation of a glutamine residue into peptide 21. Thus, the amino acid analysis supports the genetic evidence indicating that the WI and Su-2 suppressor genes are identical.
4. Discussion The RNA triplet UAG has been identified as a nonsense codon involved both with the Nl class ofphosphatase nonsense mutations (Weigert & Garen, 1965b) and with the corresponding class of amber nonsense mutations in phage T4: (Brenner, Stretton & Kaplan, 1965). The present experiments demonstrate that this triplet exhibits a remarkable range of coding specificity. In anSu - strain it behaves as a nonsense codon, whereas in an Su + strain it may code either for serine, glutamine or tyrosine, depending on which suppressor gene, Su-I, Su-2 or Su-3, is responsible for suppression (see also the accompanying report by Kaplan, Stretton & Brenner, 1965). It has recently been shown that the carrier of suppressor activity in an Suo] strain is a serine transfer-RNA molecule (Capecchi & Gussin, 1965; Wilhelm, Engelhardt, Webster, Garen & Zinder, manuscript in preparation). Thus, the coding specificity of a nonsense codon in the Su-L strain depends on the formation of a
TABLE
1
Amino acid substitutions in peptide 9 resulting from supp ression of the mutation G5 Amino ac id composition of peptide 9 Strain
Standard P+ Suppressed G5 Suppressed G5 Suppressed G5
Substitution
Suppressor gene
Su.] Su·2 Su-3
Lys
Trpt
Thr
GIy
Ala
Phe
Glu:j:
Tyr§
Serll
1·00 1·00 1·00 1·00
+ + + +
2·00 2·30 1·94 2·36
2·04 2·20
2·82 3·04 2·74 3·01
0·87 0·87 0·95 0·96
2·81 2·00
-
0·91
1·96 2·00
2·90 1·83
-
-
0·85
-
glutamine ~ serine glutam ine ~ glutamine glutam ine --+ tyrosine
The amino add analyses were performed as described in an earlier report (Weigert & Ga ren, 1965a). All results a re normalized to a lysine value of 1·00. t Tryptophan was det erm ined qualitatively by treating the fingerprint paper with a specific stain for tryptophan. :j: The amino acid analys is does not distinguish between glut a m ine and glutamic acid. T he conclusion t hat glutamine is the residue in t he standard prot ein which is affected by suppression is b ased on the elect rop hore t ic behavior of the proteins. Since the proteins obt ained from the Su-L andSu-2 suppressor strains, which h av e serine and tyrosine sub st it utions, respectively, are not elect r op horet ically different from the standard protein, it is concluded that the original amin o acid in the standard protein which is r eplaced by serine and tyrosine is glutamine rather than glutamic acid. § There was some destruction of tyrosine during hydrolysis of t he peptide, which in these analyses amounted to a bout 10%. The value in the Table has not been corrected for this loss. II There was some destruction of serine du ring hydrolysis of the peptide, amounting t o 9 % . The value in tho Table has not been corrected for this loss.
TABLE
2
Amino acid substitutions in peptide 1 resulting from suppression of the mutation H12 Amino acid composition of peptide 1 Strain
Substitution
Suppressor gene Lys'[
Trp
1·00
+
Standard P+ Suppressed H12
sa.:
1·00
Gly
Leu
0·98
0·90
0·86
1·19
Amino acid composition of peptides 1 Standard P+ Suppressed H12
Su·3
Ser
1·05
+2
Lys'[
Trp
Giy
Leu
Ala
2·00
+
1·96
2·96
2·04
2·08
2·84
2·04
2·00
tryptophan --+ serine
Tyrt 0·56
tryptophan --+ tyrosine
Peptides 1 and 2 are sufficiently separated in the fingerprint of the protein obtained from the Su-L strain to permit a separate analysis of peptide 1. However, in the protein obtained from the Su·3 strain, peptides 1 and 2 overlap and have been analyzed together (for details, see Weigert & Garen, 1965b). t Peptides 1 and 2 each contain a single lysine residue. In the analyses of peptide 1 alone, the results are normalized to a value of 1·00 for lysine. In the combined analyses of peptides 1 and 2, the results are normalized to a value of 2·00 for lysine. t The tyrosine loss during hydrolysis of the peptide in these analyses was about 23%. The value in the Table has not been corrected for this loss.
TABLE
3
Amino acid substitutions in peptide 21 resulting from suppression. of the mutation 845 Amino acid composition of peptide 21 Strain
Standard P+ Suppressed S45 Suppressed S45
Suppressor gene
Su-1
WI
Substitution Arg
Asp
1·00 1·00 1·00
2·04 2·13 2·17
Thr
3·29 3·07 3·62
Gly
2·51 2·37 1·89
The conditions for the analyses are as described in the preceding Tables.
Ala
7·05 6·6 1 7·15
Val
2·97 3·03 3·10
Leu
3·23 2·72 3·36
H is
0·99 0·75 1·07
GIu
2·14 1·25 1·97
Ser
1·85 2·80 2·05
glutamine -->- serine glutamine -->- glu t amine
SUPPRESSION OF NONSENSE MUTATIONS
527
transfer-RNA which can accept serine and insert it in the position specified by the nonsense codon. Further studies of the biochemical effects of mutations affecting suppression should provide useful information about the genetic control of coding. REFERENCES Brenner, S., Stretton, A. O. W. & Kaplan, S. (1965). Nature, 206, 994. Capecchi, M. & Gussin, G. (1965). Science, 149, 417. Gallucci, E. & Garen, A. (1966). J. Mol. Biol. in the press. Garen, A., Garen, S. & Wilhelm, R. C. (1965). J. Mol. Biol. 14, 167. Kaplan, S., Stretton, A. O. W. & Brenner, S. (1965). J. Mol. Biol. 14, 528. Notani, G. W., Engelhardt, D. L., Konigsberg, W. & Zinder, N. (1965). J. Mol. Biol.12, 439. Signer, E. R., Beckwith, J. R. & Brenner, S. (1965). J. Mol. Biol. 14, 153. Stretton, A. O. W. & Brenner, S. (1965). J. Mol. Biol. 12, 456. Weigert, M. & Garen, A. (1965a). J. Mol. Biol. 12, 448. Weigert, M. & Garen, A. (1965b). Nature, 206,992.