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Amino acid replacements resulting from super-suppression of a nonsense mutant of yeast
270
PRELIMINARY NOTES
BBA 91218
Amino acid replacements resulting from super-suppression of a nonsense mutant of yeast Genes causing the simultaneous suppression of several biosynthetically unrelated mutants of Saccharomyces cerevisiae have been termed "super-suppressors ''13. More than fifteen distinct super-suppressor genes are known and some of these have been subjected to extensive genetic studies 2,*. The results of certain genetic studies have suggested that the gene products of at least some of the super-suppressors are not proteins 4. The nature and mode of action of the super-suppressors has been inferred by indirect evidence ~ and by analogy with the bacterial nonsense suppressors a. Eight super-suppressor genes designated class I, set I, are indistinguishable by their pattern of suppression, although they are distinguishable by being not closely linked 2,*. An interaction effect has been observed wherein any combination of two of these eight genes in a haploid severely retards growth or is lethal 2. One model that has been proposed to account for these findings is that all eight genes encode the same species of transfer RNA. Mutation of any of these eight genes can cause the altered transfer RNA to translate a nonsense codon as an amino acid. In a haploid cell, modification of two or more of these transfer RNA genes, by mutation to supersuppressors, might decrease to a limiting level the amount of this transfer RNA that is available to recognize the normal codon 2. Alternatively, if the nonsense codon is used as a normal stop codon in polypeptide formation, the interaction effect could result from the super-suppressors interfering with normal polypeptide chain termination. In this paper we present direct evidence that super-suppressors do indeed act on a nonsense codon. Furthermore, in support of the above model, we show that all of the eight class I, set I, super-suppressors do cause the translation of this nonsense codon as a single amino acid, tyrosine. The genetic and biochemical techniques employed in this study may be found through the cited references. Cytochrome c-deficient mutants of S. cerevisiae are termed cy~ mutants if they carry lesions in the structural gene for iso-i-cytochrome c (ref. 6). The cyl_ 2 mutation completely prevents the synthesis of this protein. An intragenic revertant of cyl_ ~ contains iso-I-cytochrome c in which a tyrosyl residue replaces a glutamyl residue located near the site of heme attachment 7. A total of five different amino acid replacements have been found in iso-I-cytochromes c from a total of 23 intragenic revertants of the cyi_ 2 mutant**. The RNA codons assigned to these five amino acids indicate that the cyt_ 2 mutant contains the nonsense codon UAA (ochre) or UAG (amber). It appears more likely that the nonsense codon is UAA (ochre), since none of the replacements was tryptophan**. Each of the eight class I, set I, super-suppressors, when individually coupled with the cyz_o, mutant, allows the production of iso-I-cytochrome c, although in amount less than that characteristic of normal strains. The iso-I-cytochromes c have been extracted and extensively purified from eight haploid strains, each carry" D. C. HAWTHORNE AND t1. I(. MORTIMER, p e r s o n a l c o m m u n i c a t i o n s . * J. VV'. ST]~WART AND F. SItERMAN, p e r s o n a l c o m m u n i c a t i o n . Biochim. )3iophys. Acla, 161 (i968) 270-272
PRELIMINARY NOTES
ELECTROPHORESIS
271
-
ORIGI~
-~
~
ORIG~N
c s © o °O cpoO 0 4} ~ ~o@CS %0
sO
~"0 o
0
G _ CHYMOTRYPTICDIGEST
IRYPTICDIGEST
Fig, I. Tracings of peptide maps of chymotryptic and tryptic digests of iso-l-cytochromes c from n o r m a l a n d s u p p r e s s e d m u t a n t (Sk cy1_2 ) s t r a i n s . B l a c k e d in s p o t s i n d i c a t e p o s i t i o n s of v a r i a n t p e p t i d e s fro m t h e s u p p r e s s e d m u t a n t . T h e n o r m a l p e p t i d e s t h a t are m i s s i n g f r o m t h e m a p s of d i g e s t s of t h e s u p p r e s s e d m u t a n t are l i n k e d b y a r r o w s to t h e p o s i t i o n s of t h e v a r i a n t p e p t i d e s t h a t r e p l a c e t h e m . T h e p r e s e n c e of t w o h e m e p e p t i d e s in t h e m a p s of t h e t r y p t i c d i g e s t s is due to enzymic nonspecificity.
TABLE
I
AMINO ACID COMPOSITIONS SUPPRESSED
cYl_ ~ MUTANT
OF YEAST
ISO-I-CYTOCHROME
C FROM A NORMAL STRAIN AND FROM EIGHT
STRAINS
The v a l u e s r e p o r t e d are t h e a v e r a g e s of d u p l i c a t e a n a l y s e s of 2o-h, 6 M HC1 h y d r o l y s e s . The n u m b e r s of r e s i d u e s p e r m o l e c u l e of p r o t e i n w e r e c a l c u l a t e d a s s u m i n g i o o a m i n o a c i d s p e r mol e c ul e . T h i s v a l u e e x c l u d e s t h e t h r e e r e s i d u e s of h a l f - c y s t i n e a n d t h e one r e s i d u e of t r y p t o p h a n w h i c h are n o t r e c o v e r e d q u a n t i t a t i v e l y in acid h y d r o l y s a t e s a n d e x c l u d e s t h e four r e s i d u e s of prol i ne , w h o s e p e a k s are n o t r e s o l v e d f r o m c y s t e i n e .
.4 mino acid
Lysine Histidine Arginine A s p a r t i c acid Threonine Ser ine G l u t a m i c acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
Composition (residues/molec~le) Normal
Suppressed"
Normal sequence*"
16.1 3.9 3. I Ii.I 7.7 4.0 9.5 4. I 12.1 7 -1 2.5 3. I i-9 4.0 8.1 4.6 4.0 --
16.1, 3 .6 , 2.9, 11.2, 7.7, 3.8, 8.4, 4.2, 11.9, 7 .2 , 1.7, 3.°, i .7, 3.8, 7.7, 5.5, 3 .8 ,
16 4 3 ii 8 4 9 4 12 7 3 3 2 4 8 5 4 I
-
-
16. 4 3 .8 3. I 11. 5 7.9 4.2 8.6 4.7 12.2 7.4 2.4 3. I 2.0 4.0 8.1 5.7 4 .0
" M i n i m u m a n d m a x i m u m v a l u e s o b t a i n e d for t h e e i g h t i s o - i - c y t o c t l r o m e s c from t h e supp r e s s e d cyl_ 2 m u t a n t s . T h e e i g h t s u p e r - s u p p r e s s o r s are Sk, S1, Sin, Sn, So, Sp, Sq, Sr (rcf. 2 a n d D. C. HAWTHORNE AND R. K. MORTIMER, p e r s o n a l c o m m u n i c a t i o n ) . ** V a l u e s t a k e n f r o m t h e a m i n o acid s e q u e n c e r e p o r t e d b y NARITA et al. 8.
Biochim. Biophys. Acta, 161 (1968) 270-272
272
PRELIMINARY NOTES
ing a different suppressor (Sk, S1, Sm, Sn, So, Sp, Sq, Sr) and the cyi_ 2 mutant. The total amino acid composition of these proteins and the normal protein are presented in Table I. In comparison with the normal protein, there is one less residue of glutamic acid and one more residue of tyrosine in each of the proteins from the strains carrying the suppressors and the cyl~_2 mutant. Fig. I shows the peptide maps of tryptic and chymotryptic digests of isoI-cytochromes c from a normal strain and from the strain carrying the cy~_2 mutant and the suppressor Sk. Alterations are detected only in the heme peptides, and are exactly like those found in the abnormal iso-I-cytochrome c isolated from an intragenie revertant of the cyx_2 mutant which contains one tyrosyl residue replacing one glutamyl residue in the tryptic and chymotryptic heine peptides v. The suppression of the UAA or UAG codon directly demonstrates the complete functional homology between the nonsense suppressors in the procaryote bacteria and super-suppressors in the eucaryote yeast. If the super-suppressors have altered transfer RNA responsible for suppression in the same manner as the bacterial nonsense suppressors 9, then the results reported here, in conjunction with the genetic results, indicate that multiple genes encode the species of tyrosyl transfer RNA that can be made to recognize one nonsense codon as a result of a single mutational event. Whether these species are entirely alike cannot be inferred from these results. However, only one tyrosyl transfer RNA has been found in yeast and its sequence has been determined i°. This species can recognize the two tyrosine codons UAU and UAC i°. This suggests that at least eight genes encode the structure of a single tyrosyl transfer RNA. Supported by National Institutes of Health grants GM I27O2, GM-33, 39o-02, and U.S. Atomic Energy Commission (USEAC Report No. UR-49-916). We wish to acknowledge the excellent technical assistence of Mrs. N. SHIPMAN, 5{r. };'. L. X. THOMAS and Miss P. REGAN.
Department o~ Radiation Biology and Biophysics, Universit.y o~ Rochester School o~ Medicine and Dentistry, Rochesfer, N.Y. (U.S.A.)
R. A. GILMORE J . W . STE",V3.1{T I ?. SHERMAN
D. C. I-IAWTHORNE AND t{. 1(. MORTIMER, Genetics, 48 (1963) 6[ 7. I{. A. GILMORE, Genetics, 56 (1967) 641. I~. K. MORTIMER AND I~. A. GIL~IORE, ,4dvan. Biol. Med. Phys., in the press. G. E. MAGNI, R. C. VON BORSTEL AND C. M. STEINBERG, .]. Mol. Biol., 16 (1966) 568. L. GORINI AYD J. R. BECKWITH, AnSi. Rev. 3[icrobiol., 20 (1966) 4Ol. F. SHERMAN, H. TABER AND \¥. CAMPBELL, ft. 2]~Iol. Biol., 13 (1965) 21. 1?" SHERMAN, J. W. STEWART, E. MARGOLIAStt, J. PARK]ER AND \'V. CAMPBELL, Proc. Natl. Mead. Sci. U.S., 55 (~966) 1498. 8 K. NARITA, K. TITANI, Y. YAOI AND H. MURAKAMI, Biochim. Biophys. Acta, 77 (1963) 688. 9 H. M. GOODMAN, J. D. SMITH, J. N. ABELSON, A. LAND'e, F. SANGER, t3. G. BARRELL AND S. BRENNBR, Abslr. 7th Intern. Congr. Biochem., Tokyo, z967, p. 673. IO J. T. MADISON, G. A. EVERETT AND H. K. IiUNO, Cold Spring Harbor Syrup. ~)uant. Biol., 31 (1967) 409 • I 2 3 4 5 6 7
Received March i9th , I968 Biochim. Biophys. _4eta, 161 (1968) 270-272
PRELIMINARY NOTES
BBA 91218
Amino acid replacements resulting from super-suppression of a nonsense mutant of yeast Genes causing the simultaneous suppression of several biosynthetically unrelated mutants of Saccharomyces cerevisiae have been termed "super-suppressors ''13. More than fifteen distinct super-suppressor genes are known and some of these have been subjected to extensive genetic studies 2,*. The results of certain genetic studies have suggested that the gene products of at least some of the super-suppressors are not proteins 4. The nature and mode of action of the super-suppressors has been inferred by indirect evidence ~ and by analogy with the bacterial nonsense suppressors a. Eight super-suppressor genes designated class I, set I, are indistinguishable by their pattern of suppression, although they are distinguishable by being not closely linked 2,*. An interaction effect has been observed wherein any combination of two of these eight genes in a haploid severely retards growth or is lethal 2. One model that has been proposed to account for these findings is that all eight genes encode the same species of transfer RNA. Mutation of any of these eight genes can cause the altered transfer RNA to translate a nonsense codon as an amino acid. In a haploid cell, modification of two or more of these transfer RNA genes, by mutation to supersuppressors, might decrease to a limiting level the amount of this transfer RNA that is available to recognize the normal codon 2. Alternatively, if the nonsense codon is used as a normal stop codon in polypeptide formation, the interaction effect could result from the super-suppressors interfering with normal polypeptide chain termination. In this paper we present direct evidence that super-suppressors do indeed act on a nonsense codon. Furthermore, in support of the above model, we show that all of the eight class I, set I, super-suppressors do cause the translation of this nonsense codon as a single amino acid, tyrosine. The genetic and biochemical techniques employed in this study may be found through the cited references. Cytochrome c-deficient mutants of S. cerevisiae are termed cy~ mutants if they carry lesions in the structural gene for iso-i-cytochrome c (ref. 6). The cyl_ 2 mutation completely prevents the synthesis of this protein. An intragenic revertant of cyl_ ~ contains iso-I-cytochrome c in which a tyrosyl residue replaces a glutamyl residue located near the site of heme attachment 7. A total of five different amino acid replacements have been found in iso-I-cytochromes c from a total of 23 intragenic revertants of the cyi_ 2 mutant**. The RNA codons assigned to these five amino acids indicate that the cyt_ 2 mutant contains the nonsense codon UAA (ochre) or UAG (amber). It appears more likely that the nonsense codon is UAA (ochre), since none of the replacements was tryptophan**. Each of the eight class I, set I, super-suppressors, when individually coupled with the cyz_o, mutant, allows the production of iso-I-cytochrome c, although in amount less than that characteristic of normal strains. The iso-I-cytochromes c have been extracted and extensively purified from eight haploid strains, each carry" D. C. HAWTHORNE AND t1. I(. MORTIMER, p e r s o n a l c o m m u n i c a t i o n s . * J. VV'. ST]~WART AND F. SItERMAN, p e r s o n a l c o m m u n i c a t i o n . Biochim. )3iophys. Acla, 161 (i968) 270-272
PRELIMINARY NOTES
ELECTROPHORESIS
271
-
ORIGI~
-~
~
ORIG~N
c s © o °O cpoO 0 4} ~ ~o@CS %0
sO
~"0 o
0
G _ CHYMOTRYPTICDIGEST
IRYPTICDIGEST
Fig, I. Tracings of peptide maps of chymotryptic and tryptic digests of iso-l-cytochromes c from n o r m a l a n d s u p p r e s s e d m u t a n t (Sk cy1_2 ) s t r a i n s . B l a c k e d in s p o t s i n d i c a t e p o s i t i o n s of v a r i a n t p e p t i d e s fro m t h e s u p p r e s s e d m u t a n t . T h e n o r m a l p e p t i d e s t h a t are m i s s i n g f r o m t h e m a p s of d i g e s t s of t h e s u p p r e s s e d m u t a n t are l i n k e d b y a r r o w s to t h e p o s i t i o n s of t h e v a r i a n t p e p t i d e s t h a t r e p l a c e t h e m . T h e p r e s e n c e of t w o h e m e p e p t i d e s in t h e m a p s of t h e t r y p t i c d i g e s t s is due to enzymic nonspecificity.
TABLE
I
AMINO ACID COMPOSITIONS SUPPRESSED
cYl_ ~ MUTANT
OF YEAST
ISO-I-CYTOCHROME
C FROM A NORMAL STRAIN AND FROM EIGHT
STRAINS
The v a l u e s r e p o r t e d are t h e a v e r a g e s of d u p l i c a t e a n a l y s e s of 2o-h, 6 M HC1 h y d r o l y s e s . The n u m b e r s of r e s i d u e s p e r m o l e c u l e of p r o t e i n w e r e c a l c u l a t e d a s s u m i n g i o o a m i n o a c i d s p e r mol e c ul e . T h i s v a l u e e x c l u d e s t h e t h r e e r e s i d u e s of h a l f - c y s t i n e a n d t h e one r e s i d u e of t r y p t o p h a n w h i c h are n o t r e c o v e r e d q u a n t i t a t i v e l y in acid h y d r o l y s a t e s a n d e x c l u d e s t h e four r e s i d u e s of prol i ne , w h o s e p e a k s are n o t r e s o l v e d f r o m c y s t e i n e .
.4 mino acid
Lysine Histidine Arginine A s p a r t i c acid Threonine Ser ine G l u t a m i c acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
Composition (residues/molec~le) Normal
Suppressed"
Normal sequence*"
16.1 3.9 3. I Ii.I 7.7 4.0 9.5 4. I 12.1 7 -1 2.5 3. I i-9 4.0 8.1 4.6 4.0 --
16.1, 3 .6 , 2.9, 11.2, 7.7, 3.8, 8.4, 4.2, 11.9, 7 .2 , 1.7, 3.°, i .7, 3.8, 7.7, 5.5, 3 .8 ,
16 4 3 ii 8 4 9 4 12 7 3 3 2 4 8 5 4 I
-
-
16. 4 3 .8 3. I 11. 5 7.9 4.2 8.6 4.7 12.2 7.4 2.4 3. I 2.0 4.0 8.1 5.7 4 .0
" M i n i m u m a n d m a x i m u m v a l u e s o b t a i n e d for t h e e i g h t i s o - i - c y t o c t l r o m e s c from t h e supp r e s s e d cyl_ 2 m u t a n t s . T h e e i g h t s u p e r - s u p p r e s s o r s are Sk, S1, Sin, Sn, So, Sp, Sq, Sr (rcf. 2 a n d D. C. HAWTHORNE AND R. K. MORTIMER, p e r s o n a l c o m m u n i c a t i o n ) . ** V a l u e s t a k e n f r o m t h e a m i n o acid s e q u e n c e r e p o r t e d b y NARITA et al. 8.
Biochim. Biophys. Acta, 161 (1968) 270-272
272
PRELIMINARY NOTES
ing a different suppressor (Sk, S1, Sm, Sn, So, Sp, Sq, Sr) and the cyi_ 2 mutant. The total amino acid composition of these proteins and the normal protein are presented in Table I. In comparison with the normal protein, there is one less residue of glutamic acid and one more residue of tyrosine in each of the proteins from the strains carrying the suppressors and the cyl~_2 mutant. Fig. I shows the peptide maps of tryptic and chymotryptic digests of isoI-cytochromes c from a normal strain and from the strain carrying the cy~_2 mutant and the suppressor Sk. Alterations are detected only in the heme peptides, and are exactly like those found in the abnormal iso-I-cytochrome c isolated from an intragenie revertant of the cyx_2 mutant which contains one tyrosyl residue replacing one glutamyl residue in the tryptic and chymotryptic heine peptides v. The suppression of the UAA or UAG codon directly demonstrates the complete functional homology between the nonsense suppressors in the procaryote bacteria and super-suppressors in the eucaryote yeast. If the super-suppressors have altered transfer RNA responsible for suppression in the same manner as the bacterial nonsense suppressors 9, then the results reported here, in conjunction with the genetic results, indicate that multiple genes encode the species of tyrosyl transfer RNA that can be made to recognize one nonsense codon as a result of a single mutational event. Whether these species are entirely alike cannot be inferred from these results. However, only one tyrosyl transfer RNA has been found in yeast and its sequence has been determined i°. This species can recognize the two tyrosine codons UAU and UAC i°. This suggests that at least eight genes encode the structure of a single tyrosyl transfer RNA. Supported by National Institutes of Health grants GM I27O2, GM-33, 39o-02, and U.S. Atomic Energy Commission (USEAC Report No. UR-49-916). We wish to acknowledge the excellent technical assistence of Mrs. N. SHIPMAN, 5{r. };'. L. X. THOMAS and Miss P. REGAN.
Department o~ Radiation Biology and Biophysics, Universit.y o~ Rochester School o~ Medicine and Dentistry, Rochesfer, N.Y. (U.S.A.)
R. A. GILMORE J . W . STE",V3.1{T I ?. SHERMAN
D. C. I-IAWTHORNE AND t{. 1(. MORTIMER, Genetics, 48 (1963) 6[ 7. I{. A. GILMORE, Genetics, 56 (1967) 641. I~. K. MORTIMER AND I~. A. GIL~IORE, ,4dvan. Biol. Med. Phys., in the press. G. E. MAGNI, R. C. VON BORSTEL AND C. M. STEINBERG, .]. Mol. Biol., 16 (1966) 568. L. GORINI AYD J. R. BECKWITH, AnSi. Rev. 3[icrobiol., 20 (1966) 4Ol. F. SHERMAN, H. TABER AND \¥. CAMPBELL, ft. 2]~Iol. Biol., 13 (1965) 21. 1?" SHERMAN, J. W. STEWART, E. MARGOLIAStt, J. PARK]ER AND \'V. CAMPBELL, Proc. Natl. Mead. Sci. U.S., 55 (~966) 1498. 8 K. NARITA, K. TITANI, Y. YAOI AND H. MURAKAMI, Biochim. Biophys. Acta, 77 (1963) 688. 9 H. M. GOODMAN, J. D. SMITH, J. N. ABELSON, A. LAND'e, F. SANGER, t3. G. BARRELL AND S. BRENNBR, Abslr. 7th Intern. Congr. Biochem., Tokyo, z967, p. 673. IO J. T. MADISON, G. A. EVERETT AND H. K. IiUNO, Cold Spring Harbor Syrup. ~)uant. Biol., 31 (1967) 409 • I 2 3 4 5 6 7
Received March i9th , I968 Biochim. Biophys. _4eta, 161 (1968) 270-272