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Demonstration of the UAA ochre codon in bakers' yeast by amino-acid replacements in iso-1-cytochrome c
J. Mol. Bid.
(1972) 68, 8346
Demonstration of the UAA Ochre Codon in Bakers’ Yeast by Amino-acid Replacements in Iso-l-cytochrome c JOHN W. STEWART,FBED FRED L.X.THolldas
SHEBMAN,~XARYJA~KSON ANDNANCYSHIPMAN
of Radiation Biology and Biophysics University of RochesterSchool of Medicine and Dentistry Rocheder, New York 14642, U.S.A. Department
(Received13 December1971) Iso-1-cytochromes c from 45 intragenic rev&ants of the cyl-9 mutant of bakers’ yeast carry at position 2 one of the following six amino acids: glutamic acid, glutamine, serine, tyrosine, leucine and lysine. The replacement amino acids and the genetic code identify the cyl-9 lesion ae a base-pair substitution creating UAA at the messenger RNA template for position 2. Since, in addition, the cyl-9 mutant lacks iso-1-cyt~chrome c but can be suppressed by super-suppressor genes, this UAA triplet is a nonsense codon in yeast. The absence of other amino acids among the replacements suggests that UGA also is a nonsense codon in yeast. When acting to revert the ~~1-9 U&4 nonsense lesion, X-rays, some alkylating chemicals and spontaneous events cause substitutions of A*T base pairs with no apparent selectivity, whereas ultraviolet irradiation and nitrous acid selectively induce A*T-+ G-C. Single substitutions account for all 45 reversions either obtained spontaneously or induced by eight different mutagenic treatments. Reinitiation past the nonsense codon at position 2 fails to occur at a detectable level, although an efficient translation initiation site is accessible by mutation of the AAG, lysine 4 codon to AUG, in other mutant strains that lack the normal initiation codon.
1. Introduction Nonsensecodons were l3rst recognized in certain rII B mutants of bacteriophage T4 (Benzer & Champe, 1962 ; Champe & Benzer, 1962). Because they could be reverted by a base analog, they were believed) to be base-substitution mutants. Since they were suppressed in certain host mutants, they were believed to generate defects that could be repaired during translation of messengerRNA. The sites occupied by the mutants couldcontrol unessential regions of a protein, since some were located in a region of the B cistron that could be bounded by certain frameshift mutations or be deleted sltogether without deleterious effect, and since others fell in the A cistron, but were expressed as rI.l B mutants when the two cistrons were fused by deletions. Only if these mutations had generated coding units that did not correspond to any amino acid could these circumstances be readily understood. The different pattern of suppressionof nonsensemutations led to the distinction of amber and ochre mutants (Brenner & Beckwith, 1965). Amino-acid replacements in revertants of ember mutants of the structural genefor alkaline phosphatesein Escherichiu coli were found restricted to those having codonsdiffering by one basefrom UAG, 83
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and this coincidence was understood to imply that UAG is the non-coding, amber triplet (Weigert & Garen, 1965). In this way also, the ochre nonsensecodon was identified as UAA (Weigert, Lanka, & Garen, 1967). A third suppressible nonsense triplet has been assignedthe identity UGA by the use of mutagens having known modes of action (Brenner, Barnett, Katz t Crick, 1967). Bacterial suppressors specific for UGA, UAG, or both UAG and UAA, suppressedall 14 different nontemperature-sensitive, base analog mutants detected in comprehensive mutational studies of the expendable region of the rII B cistron, and they also suppressedsix of the seven carefully analyzed barriers to reading frameshifts in this region (Barnett, Brenner, Crick, Sohulman & Watts-Tobin, 1967). Furthermore, only these three triplets remain unassigned as amino-acid codons by in vitro studies of the coding properties of all 64 nucleotide triplets (reviewed by YEas, 1969). UAA, UAG and UGA therefore are evidently the only nonsensecodonsin E. coli. Mutational introduction of the amber triplet into the normal reading frame (Brenner & Stretton, 1965) brings about the termination of growth of nascent polypeptide chains, at the residue coded by the triplet just preceding UAG (Stretton & Brenner, 1965), and causestheir release(Sarabhai, Stretton, Brenner & Bolle, 1964). The ochre codon UAA occupies the site expected for a natural chain termination codon in the coat protein cistron of bacteriophage R17 (Nichols, 1970). All three nonsensecodons signal the release of growing pepticles from peptidyl-tRNA on cell-free ribosomesof E. coli (Caskey, Tompkins, Scolnick, Caryk & Nirenberg, 1968). The three nonsense triplets, individually or in tandem combination (Nichols, 1970; Lu & Rich, 1971), are apparently the normal signals for chain termination in E. di. Studies with eucaryotic cell-free preparations have left UAA and UAG among the eight triplets not yet assignedas amino-acid codons (Marshall, Caskey t Nirenberg, 1967; S6ll, Cherayil & Bock, 1967; Caskey, Beauclet & Nirenberg, 1968; Gupta, 1968), and suggestthat the three bacterial nonsensetriplets can signal the releaseof pepticles from peptidyl-tRNA in mammalian cells (Beaudet & Caskey, 1971). It would be reassuring to find that these three triplets are in fact nonsense codons in living eucaryotic cells. In this paper we show that UAA is a nonsensecodon in bakers’ yeast. Amino-acid substitutions in iso-1-cytochrome c from revertants of the cyl-9 mutant identify its lesion as UAA replacing the GAA triplet that normally encodesglutamic actid2 in the protein. The cyl-9 mutation prevents the occurrence of the protein, although residue 2 may be varied greatly or deleted altogether without adverse effect. Completing the parallel with nonsensemutants in E. co&, the cyl-9 mutant is suppressible(Gilmore, Stewart & Sherman, 1971).
2. Materials and Methods (a) Genetie nmmlatolre
and strain.9
The symbol CYl denotes the wild-type structural gene for iso-1-cytoohromec; cgll denotesmutants of this gene, which are substantially deficient in amount or activity of iso-1.cytochrome c. Allele numbers following the cyl symbol, as in cyl-9, distinguish mutants derived independently. Intragenic revertants are designated by CYl followed by the allele number of the cyl parent strain, followed in turn by allele letters to distinguish independent revert&nts. For example, CYl-9-A and CYI-9-B designate two intragenic revertants of ql-9. The original cyl-9 mutant was induced by ultraviolet irradiation and was detected by the benzidine staining method (Sherman et al., 1968). The two haploid strains used in this
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study, JP109-3A and JP109-6A, are segregants having the genotype OL ~~1-9 p+, which were obtained from a cross of the origi.nal cyl-9 p- strain with a normal strain (Parker & Sherman, 1969). Independent revertants were selected on k&ate medium (Sherman et al., 1968) after no treatment or after treatments of either 1.5 krad. of X-rays or 1600 ergs mmma of ultreviolet irradiation, or 15 mm with 50 mM-nitrous acid, or 30 mm with 3 ma6-l-nitrosoimidazolidone-2 (Sherman et al., 1968; Stewart, Sherman, Shipman & Jackson, 1971; Parker 6t Sherman, 1969), or 40 min with 2% (v/v) ethyl methanesulfonate. Revertants were obtained with diethylsulfate, N-methyl-N’-mtro-N-nitrosoguanidine and methyl methanesulfonate by streaking a diluted cell suspension on glycerol medium (1% Bscto-yeast extract, 2% Bacto peptone, 3% (v/v) glycerol and 2% agar), and placing a paper disc with the mutagen on the surface of the plate. The discs were removed after 2 days of incubation and the plates were replica plated onto lactate medium. Over 300 revertants were purified by subcloning and the subolones were examined for their cytochrome content by lowtemperature spectroscopy. Forty-six revertants with approximately normal amounts of cytochrome c, judged by spectroscopy of whole cells, were chosen for this study. Forty of these were analyzed genetically to determine whether the back mutation was at the cyl locus (Sherman et al., 1968). The revertants were crossed to normal haploid strains and 20 segregants from each of the sporulated diploids were examined for their cytochrome spectra. (b)
Growth
of yea&
and
preparation
of iso-1-cytochrome
c
Ten-liter batches of the cyl-9 revertants were grown as described previously (Stewart et al., 1971), yielding from 0.5 to 1.0 kg of wet cells. Iso-1-cytochrome c was extracted and purified by the methods of Sherman et al. (1968). To achieve adequate purity in a few instances, a repetition of the chromatographic purification on CG50 cation-exchange resin (200 to 400 mesh) was required. Yields of iso-1-cytochrome c were 20 to 90 mg /lO-l.batch. (c) Identijicution
of structural
changes
in iso-1-cytochrome
c
enzymic digestion, peptide mapping, The methods used for amino-acid analysis, sequential Edman degradation and chromatographic identification of the phenylthiohydantoins have been described (Stewart et al., 1971). Proteins were hydrolyzed in constsntboiling hydrochloric acid in sealed, evacuated tubes at 111°C for 20 hr, and the hydrolysates were analyzed with a Beckman-Spinco amino-acid analyzer, model 120C. Molar compositions were based on a theoretical content of 79 acidic and neutral amino-acid residues, other then cystine and methionine, and 23 basic amino-acid residues per molecule of protein (Narita & Titani, 1969), except in those instances when 78 and 24 gave more acceptable results. Tryptic and chymotryptic digests of Bamples of 0.1 pmole of oytochrome c were prepared by 2.75 hr of reaction at 37°C of 1% w/v cytochrome c and 0.1% w/v enzyme in 0.25% w/v ammonium bicarbonate. The digests were lyophylized and were subjected to electrophoresis on Whatman no. 3 MM paper wet with pyridine/acetic acid/water (5:0.18: 95), pH 6.5, for 1.66 hr at 20 V/cm, and then to descending chromatography in n-butanol/pyridine/acetic acid/water (15: 10: 3 : 12) for 16 hr. The peptide maps were developed by sequential dipping through the collidine/ninhydrin reagent, the Ehrlich reagent for tryptophan, and the Pauli reagent for histidine and tyrosine, in that order. Samples of 0.3 pmole of cytocbrome c were subjected to sequential Edman degrsdation and the phenylthiohydantoins were identified by thin-layer chromatography on silica gel.
3. Results The cyl-9 mutant was recognized as possibly a nonsense mutant in the course of a program of amino-acid analysis and peptide mapping of iso-I-cytochromes c from revertants of cyl mutants which were altered in unknown ways (for example, 888 Sherman et al., 1968). Spectroscopic examinations of whole ceIIs of cyl-9 strains and growth tests on lactate medium indicated a level of total cytochrome c equal to no more than the normally small content of iso-2-cytochrome c. The amino-acid composition of iso-l-cytoohrome c from rev&ant C yl-9-C appeared unaltered, but normal
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peptides C-l and T-l were missing from the chymotryptic and tryptic digests, respectively, and each was replaced with a more basic peptide (Sherman et al., 1963). These analytical results, when viewed in the knowledge that C-l and T-l are the NH,-terminal peptides Thr-Glu-Phe and Thr-Glu-Phe-Lys (Stewart et al., 1971), suggestedthat glutamic acid 2 may have been replaced by glutamine. Since codons for glutamic acid and glutamine are related to the ochre and amber nonsensetriplets by single basechanges(seeYEas, 1969), these observations prompted a detailed examination of the iso-1-cytochrome c content of cyl-9, and of the structural changesin iso-1-cytochromes c from many cyl-9 revertants. (6) Absenceof iso-f-cytochrome c in cyl-9 Cytochrome c was extracted from the cyl-9 mutant JPIOS-BA and was chromatographed on cation-exchange resin. The cytochrome c eluted at a single position characteristic of iso-2-cytochrome c. The total yield was approximately 2.5 mg from an amount of yeast corresponding to 0.44 kg dry wt. This yield is within the normal range of iso-2-cytochrome c concentrations, 6 to 30 mg/kg dry weight (Sherman & Stewart, 1971; Gilmore et al., 1971; Stewart et al., 1971). No other chromatographic zone of cytochrome c was observed, although a zone of iso-1-cytochrome c equal to somewhat lessthan 10 o/oof this amount of iso-2-cytochrome c would have been seen. Since the level of iso-2-cytochrome c in both normal and cyl mutant strains is usually slightly lessthan 5% of the level of iso-1-cytochrome c in normal strains (Sherman, Taber t Campbell, 1965; Gihnore et al., 1971; Sherman t Stewart, 1971; Stewart et aZ., 1971), this result fixes a conservative upper limit of iso-1-cytochrome c content in JP109-6A at 0.5% of the normal amount. (b) Iso-I-cytochromes c in revertants of cyl-9 (i) Selection of revertunts Over 300 revertants that exhibited approximately normal growth on lactate medium were selected after treatments of ~~1-9 strains with various mutagens and after no treatment. Normal growth responseindicates the presenceof more than 10% of the normal total cytochrome c activity for lactate utilization (Gilmore et al., 1971). Almost all these revertants revealed approximately normal intensities of the cytochrome a-bands. The cytochrome a-band intensities provide a sensitive assay of total cytochrome c content: for example, spectroscopic estimates of 50% normal levels in whole cells were confirmed by extraction procedures (Stewart et al., 1971). Forty revertants containing normal amounts of cytochrome c were tested genetically for closelinkage between the reversion site and the cyl-9 site. One revertant, B-929, was clearly due to s mutation at a gene distant from cyl, but the other 39 strains appeared due to intragenic reversion. The exceptional strain, B-929, yielded a normal amount of total cytochrome c, which was entirely iso-2-cytochrome c on the basisof preparative chromatographic results, peptide maps and amino-acid composition. Closerscrutiny of the low-temperature spectrum of whole cells of this strain revealed an unusually narrow c,-band that was shifted slightly towards the blue. This abnormal spectroscopic behavior might have been anticipated as an indication of iso-2cytochrome c, since low-temperature spectrophotometric tracings of purified cytochromesc show that the c,-band of iso-2cytochrome c is slightly narrower than that of iso-1-cytochrome c and is shifted O-5 nm toward lower wavelengths (Sherman 8r Stewart, 1971). In similar studies with other cyl mutants, we have found (Sherman &
UAA
OCHRE
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Stewart, unpublished experiments) that comparably small proportions of the revertants are due to extragenic suppressorswhich elevate the content of iso-2-cytochrome c to a level normally found for iso-1-cytochrome c. In all cases, these revertants could be recognized by their narrow and shifted c,-band. On the other hand, iso-l-cytochrome c was obtained from all extracted revertants that showed the normally intense but normally broad c,-band in spectra of whole cells, and genetic tests of these strains have always indicated that they were due to an intragenic reversion event (for example, seeSherman et al., 1968; Stewart et al., 1971). These observations indicate that revertants exhibiting normal spectra, with respect to the width and exact position as well as the relative intensity of the c,-band, are in all likelihood intragenic revertants. The 39 intragenic revertants that were tested genetically, as well as the six X-ray-induced revertants that contained normal c,-bands and normal amounts of iso-1-cytochrome c, are listed in Table 1. (ii) Iso-I-cytochromes c Approximately normal yields of iso-l-cytochrome c were obtained from all 45 revertant strains in Table 1. Amino-acid compositions of iso-l-cytochromes c in the 45 revertants are listed in Table 2. Because only five different types of composition were apparent, only the averages of all compositions judged to be identical are given. The five compositional types include the normal and four abnormal ones,which differ from the normal by the loss of one residue of glutamic acid and by the gain of one residue of either lysine, serine, leucine or tyrosine per molecule of protein. To indicate the reliability of the assignmentsof the 45 individual analysesto the five groups, also listed in Table 2 are the ranges of the extreme values for each amino-acid residue for all 45 proteins, normalized by the appropriate addition of 1.00 residue of glutamic acid and subtraction of l-00 residue of the extra amino acid. The integral compositional changes of the iso-1-cytochromes c in all 45 revertants are listed in Table 1. Peptide maps of all 45 proteins are summarized in Figure 1. Six different types of tryptic and chymotryptic peptide maps were found. They distinguish the samefive sets of proteins as the compositions, but subdivide the proteins having normal compositions into a classhaving normal peptide maps and a classhaving altered peptide maps. All of the abnormal peptide maps lack spots C-l and T-l but contain all of the other normal spots, as well as extra spots, which permit the distinction of the five altered types. The missing spots C-l and T-l are the NH,-terminal chymotryptic and tryptic peptides, Thr-Glu-Phe and Thr-Glu-Phe-Lys, respectively (Stewart et al., 1971). The extra spots for all 45 proteins are listed in Table 1. The net changesin composition and in peptide maps of all 45 iso-l-cytochromcs c are grossly consistent with the replacement of glutamic acid 2 with any of six different amino acids, including glutamine and glutamic acid. The changesin the peptide maps may be examined in more detail, to test this suggestion. The new peptides in the chymotryptic digests of all 44 altered proteins, except those containing extra lysine, are electrophoretically neutral at pH 6.4, whereasthe new chymotryptic peptide from the proteins having extra lysine exhibits a mobility towards the cathode approximately equal in magnitude to the mobility of peptide C-l, Thr-Glu-Phe, towards the anode. Electrophoretically, these changesare consistent with replacement of glutamic acid 2 in Thr-Glu-Phe with neutral amino acids including glutamine, and with lysine. Chromatographically, the new chymotryptic peptides are related to Thr-Glu-Phe in
88
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TABLE
ET
AL.
1
Alterations of iso-1-cytochrm c in 45 inbragenicrevertants of cyl-9 straimt Rev&ant
strain
Alterations
Number
Genotype
Mutagen%
B-496 B-496 B-497 B-829 B-830 B-831 B-832 B-833 B-834 B-835 B-836 B-837 B-913 B-914 B-915 B-916 B-917 B-918 B-919 B-921 B-922 B-923 B-924 B-926 B-1038 B-1080 B-1081 B-1082 B-1089 B-1090 B-1091 B-1098 B-1099 B-1100 B-1290 B-1291 B-1959 B-1960 B-1961 B-1962 B-1963 B-1964 B-2069 B-2070 B-2071
CYl-9-A CYl-9-B GYl-9-C CYl-9-D CYl-9-E CYl-9-F CYl-9-G CYl-9-H CYl-9-I CYl-9-J CYl-9-K CYl-9-L CYl-9-M CYl-9-N CYl-9-o CYl-9-P CYl-9-Q CYl-9-R CYl-9-s CYl-9-T CYl-9-u CYl-9-v CYl-9-w CYl-9-x CYl-9-Y CYl-9-z CYl-g-AA CYl-9-AB CYl-O-AC CYl-g-AD CYl-9-AE CYl-9-AF CYl-9-AG CYl-S-AH CYl-9-AI CYl-9-AJ CYl-B-AK CYl-g-AL CYl-O-AM CYl-g-AN CYl-g-A0 CYl-9-AP CYl-9-A& CYl-g-AR CYl-O-AS
U.V. U.V. . . Gale U.V. U.V. U.V. U.V. U.V. U.V. U.V. U.V. None EMS EMS EMS EMS NIL NIL NA NA NA NA NA None DES DES DES NG NC NG MMS MMS MMS NIL NIL X-ray X-ray X-ray X-ray X-ray X-ray None None None
Change in composition$ YMidueal~OleCulS None None None None None None None None None None None None None None None -Glu, +Lys -Glu, +Leu - Glu, +Lys -Glu, +Tyr None None - Glu, +Leu None None -Glu, +Tyr - Glu, +Lys -Glu, +Ser None None None -Glu, +Lys -Glu, +Tyr - Glu, +Tyr -Glu, +Leu None -Glu, +Lys - Glu, +Ser - Glu, +Tyr - Glu, +Tyr - Glu, +Lys None - Glu, + Leu -Glu, +Ser None -Glu, +Tyr
Peptide map7 replacement of C-l and T-l b b b b b b b b b b b b b b b a e d” b b b b d a E b b 1 d i a d d b” e c None
d
Replacement of glut’amic acid 2
GinII Gln Gln Gln Gln Gln Gln Gln Gln Ghl Gln Gln Gln Gln Gln LYS Leu LYS TV Gln Gln Leu Gln Gln TF LYS SW! Gln Ghl Ghl LYS Tyr TV L&l Gln Lys Ser TF Tyr LYS Ghl Leu Ser Glu TF
t The cyl-9 strain that was revert-ad to produce revertant strains B-496, B-496 and B-497 was JP109-3A; the cyl-9 strain from which all other revertants were prepared was JP109-6A. $ The abbreviations of mutagens used are as follows: u.v., ultraviolet light; EMS, ethyl methanesulfonate; NIL, 1-nitrosoimidazolidone-2; NA, nitrous aoid; DES, diethyl sulfate; NC, iv-methylN’.nitro-N-nitrosoguanidine; MMS, methyl methanesulfonate. $ Amino-acid compositions of the proteins are in Table 2. 1 Peptide maps of chymotryptic and tryptic digests are in Fig. 1. jl Glutamine was directly shown, by chromatography of the phenylthiohydantoin of residue 2, only for CYl-9-A and CYl-9-B; in all other cases it was inferred from the neutrality, at pH 6.4, of the replacement of Thr-Glu-Phe and from the normal amino-acid composition.
1
TryptoPh
2
Type
of composition
6
1
1'7.26 3.68 3.06 11.13 7.65 4.10 8-64 4.37 II-85 7.24 1.82 3.06 1.82 3.81 7.64 4.72 3.89
-Glu,+Lys
4
1
2.26 3.06 1.74 3.79 8.74 4.73 3.90
11.90 7.18
4.01 8.48 4.39
11.18 7.61
16.15 3.86 2.99
Average
- Glu, + Ser
3
1
3.90 2.99 Il.33 7-72 4-80 8-47 4.21 11.92 7.16 2.66 3.02 l-63 3.89 7.86 4-65 3.97
16.11
residues/molecule
- Glu, +Leu
Range
16.16 3-80 3.04 11.20 7-65 4.07 9.63 4.29 Il.89 7.34 2.19 3.04 1.78 3.78 7.79 4.63 3.89
Midpoint
7xJO 1.16 2.93 1.38 3.55 7.56 4.35 3.64
11.51
16-89 3.52 2,87 10.86 7.47 3.76 9.26 3-88
of 46 normalized Minimum
16.42 4.08 3.20 11.84 7.83 4.39 9.81 4.70 12.27 7-67 3.22 3.16 2.09 4.00 8.02 4.91 4.14
values ilfaximum
Results are from single analyses of 20-hr hydrolysates of each protein in 6 N-HCl at 11 l”C, for all residues except tryptophan; values for tryptophan are based on results of staining peptide maps with an Ehrlich reagent. Molar values were calculated from mole fraotions, assuming 79 neutral and acidic residues and 23 basic residues, excluding tryptophan, oystine and methionine, per mole of normal protein; 78 and 24 were assumed instead for the - Glu, +Lys proteins. Values reported are averages of values for all proteins judged identical; the number of protein analyses averaged is shown at the bottom of each column. To obtain a single measure of dispersion of the values for all 46 proteins, the analysis of each protein was either left unaltered or adjusted to the normal by addition of I.00 residue of glutamio said and subtraction of I.00 residue of tyrosine, Iysine, leuome, or serine, as diotated by the change of its compositional type from the normal; the extremes of these 45 normalized values and the midpoints of these extremes are recorded.
Total
25
Phenylalanine
proteins
7.23 2.26 3.05 1.83 3.79 7.79 5.72 3.86 1 7
TyrOShlt3
11.96
16.20 3.79 3.01 Il.17 7.72 4.05 8.66 4.11
- Glu, f Tyr
7.22 I.98 3.04 1.85 3.86 7.89 4.74 3.92
7.66 3.94 9.48 4.28
11.10
3-77 3.02
1621
Normal
11.91
acid
acid
acid
Glycine Allmine Half-cystine Vsline Methionine Isoleuoine Leucine
PrOline
LyfJin0 Hi&dine Arginine Aspartio Threonine serine Glutamio
Amino
TABLE
Amino-acid composition of 45 iso-1-cytochromesc
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Electrophoresis
I -: Origin I
ET
Orlgln J
Chymotryptlc
:I
Origfn J
digest
Tryptic
digest
FIQ. 1. Cumulative peptide maps of 45 iso-1-cytochromes c. Outlined areas represent nermal spots on ninhydrin-stained peptide maps of iso-1-cytochrome c from wild-type bakers’ yeast and from revertant strain B-2070 (CYl-S-AR). C-l and T-l are normal NH,-terminal peptides and are missing from peptide maps of all revertant strains except B-2070. (0) Mark centers of new, ninhydrin-positive peptides of &ered proteins. Lower case letters designate amino-acid replaaemerits of glutamic acid in proteins yielding these new peptides, as follows: lysine, a; glutamine, 5; se&e, c; tyrosine, d; and leucine, e. In all 90 peptide maps, spots at normal positions gave normal color responses with the oollidine-ninhydrin, Ehrlich and Pauli reagents. All new spots except the lower of the tryptio peptides a gave yellow colors initielly with the ninhydrin reagent. All new spots gave negative Ehrlich color reactions for typtophan, and all new spots except d, which were tyrosine-positive, failed to react positively with the Peuli reagent. Left panel shows a chromatogram of amino acids, applied to a peptide map at the same height as visible heme peptides after electrophoresis but before chromatography. Numbers designate amino acids as follows: lysine, 1; glutamine, 2; glutamic acid, 3; serine, 4; tyrosine, 5; phenylalanine and leutine. 0.
much the same ways as the corresponding amino-acid replacements are related to glutamic acid, as shown in the left panel of Figure 1, with one exception. The single exception is the uppermost of the new peptides d in chymotryptic digests of proteins having extra tyrosine; it is relatively too high on the chromatogram. The yield of this upper peptide d is low, since it gives a relatively faint ninhydrin spot and is revealed primarily by its slightly positive response to the Pauli reagent ; in contrast, the lower peptide d is strongly responsive to both reagents. The upper peptide d may be Thr-Tyr, which could result from chymotryptic digestion of Thr-Tyr-Phe; the confirmatory presence of phenylalanine was not detected, but small amounts of phenylalanine would be masked by normal spots. Also, it could result from digestion of larger NH,-terminal fragments. Similar analysis of the tryptic peptide maps also permits the conclusion that neutral
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residues replace glutamic acid 2 in Thr-Glu-Phe-Lys with the exception of the apparently normal protein in CYl-9-AR and the proteins having extra Iysine. The tryptio digest8 of proteins having glutamic acid replaced by lysine contain two new peptides, a in Figure 1, which are both strongly responsive to the ninhydrin reagent. Both are more mobile toward8 the cathode than the other new peptides. These two peptides a are plausibly Thr-Lys and Phe-Lys, the expected product8 of tryptic digestion of Thr-Lys-Phe-Lys. The uppermost tryptic peptide a may be Thr-Lys, for it gave a yellow color initially during reaction with the ninhydrin reagent; in our experience yellow color8 are given by peptides only when they carry NH,-terminal residues of threonine, serine, glycine or asparagine. It is significant that all of the new spots, in both enzymic digests, except for the lower of the new tryptic peptides a from the proteins having extra lysine, gave an initial yellow color during reaction with the ninhydrin reagent. This result indicate8 a retention of threonine at the NH,-terminus of all the proteins. Retention of phenylalanine at position 3 is suggested by the chymotryptic peptide maps, to account for the universal presence of peptide C-2, which includes residue8 4-14 (Stewart et al., 1971). These detailed examinations of the peptide map8 tend to 6x the site altered as position 2. The amino-acid sequence of the three NH,-terminal residues of the iso-l-cytochromes c from the first two revertants GYl-9-A and CYl-9-B was determined by sequential Edman degradation. The NH,-terminal sequence of both proteins was Thr-Gln-Phe.
4. Discussion (a) The ~~1-9 mutation is a nonsense mutation The drop in iso-l-cytochrome c content to below 1% of the normal level in strain JP109-6A reveal8 substantial unacceptability of the cyl-9 mutational lesion. The revertability of the lesion with a variety of mutagens suggests that the mutation is due to a base-pair substitution rather than deletion or insertion of base pairs. This suggestion is supported by the restriction of the site of primary structural alterations in the 45 revertant iso-l-cytochromes c to a single residue position. Furthermore, the aminoacid replacement8 observed are consistent with single base-pair substitution in the cyl-9 mutation, as will be detailed in section (b) below. The lesion probably does not prevent the formation of mRNA, since it is weakly suppressed by class 1, set 1 supersuppressors, which may act through altered transferRNA’s (Gilmore et al., 1971). The cyl-9 mutation is apparently a substitution mutation that prevent8 translation or causes gross instability of iso-l-cytochrome c. A single amino-acid change at position 2 that causes gross abnormality of the protein seems an unlikely explanation for the deficiency in cyl-9, since the replacements in the revertants, glutamic acid, lysine, glutamine, serine, tyrosine and leucine, cover most of the range of structural diversity of amino acids. Furthermore, position 2 is excluded altogether in a short form of iso-l-cytochrome c, which lacks the normal, first four NH,-terminal residues, without serious impairment of function (Stewart et al., 1971). Nevertheless, certain residues at position 2 might act in especially deleterious ways. Only three such amino acid8 come to mind. Cysteine might interfere with heme addition to cysteinyls 19 and 22 in the apoprotein, or might react adversely with the single cysteinyl residue at position 107. However, cysteine does satisfactorily occupy the nearby homologous position 7 in a revertant strain B-2178 prepared from
92
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ET
AL.
~~1-183 (Stewart & Sherman, unpublished data). Methionine or histidine might provide incorrect ligands to the heme iron, but methionine satisfactorily occupies a neighboring position, at the NH,-terminus of iso-l-cytochrome c in revertants of initiator mutants (Stewart et al., 1971). If the cyl-9 mutation is in fact a single basepair change affecting the triplet encoding position 2, then it must be consideredin all likelihood a nonsensecodon. (b) The nonsense codon in cyl-9 is UAA Only one residue position appears altered in all 45 revertant proteins. Position 2, which is normally glutamic acid, has become glutamine, lysine, leucine, serine, or tyrosine, or remained glutamic acid. According to the bacterial genetic code (see Y&s, 1969), these amino acids constitute the entire set of amino acids that have codons that differ from UAA by one base change, and are all but one of the amino acids that have codonsthat differ from UAG by one basechange. They could all arise from UAPu in cyl-9 by a single base change. In contrast, instances of multiple base changeswould be required to generate codonsfor all these amino acids from any other triplets. This correlation identifies the altered messengerRNA triplet corresponding to position 2 in cyl-9 as UAPu. The cyl-9 mutation was apparently a G-C-+ T.A substitution affecting the first base of the codon for glutamic acid 2, GAPu. Complete identification of the UAPu triplet depends upon negative information. Tryptophan is not among the 45 amino-acid replacements at position 2. The UGG tryptophan codon in yeast (Stewart & Sherman, manuscript in preparation), may be derived from UAG by an A-T --+ G*C transition at the second base position. This change probably occurred at a detectable frequency, since the A-T -+ G*C transition at the iirst base,which generated the CAPu codonsfor the 24 glutamine replacements, was the preponderant changedetected, and occurred spontaneously and with seven of the eight mutagens; and since, in addition, the second baseof UAPu appearsto be as mutable as the first and third bases,judged by the frequencies of transversions (see Table 3, in section (d)). The structural variations observed at and near position 2 leave little doubt that tryptophan would be acceptable at position 2 ; substantial reduction in function would be required to preclude selection, since a 90% decreasein iso-lcytochrome c content affects only slightly the growth on lactate medium (Gilmore et al., 1971). For these reasons, the absence of tryptophan from the 45 replacements suggeststhat the UAPu triplet in cyl-9 is not UAG. Supporting this conclusion is the observation that an efficient, tryosine-inserting super-suppressorof the UAG nonsense triplet in cyl-179 (Sherman, Liebmen, Stewart dzJackson, manuscript in preparation) fails to suppress cyl-9, although other less efficient super-suppressorsthat insert tyrosine do suppresscyl-9 (Gilmore et al., 1971). By difference, the unacceptable UAPu lesion in cyl-9 is UAA. Just asin E. coti, UAA is a nonsensecodon in yeast. (c) Coding by the triplet UGA Reasonsheve been given for believing that any amino acid should be acceptable at position 2, and that the transition A*T -+ G-C affecting the secondbase of UAA in cyl-9 would have been registered among the 45 revertants if it could produce an acceptable amino acid codon. For those reasons,the UGA triplet resulting from this transition must be either a new codon for one of the amino-acid replacements, glutamic acid, lysine, glutamine, leucine, serine or tyrosine; or it must be a nonsenseoodon. A definitive choice cannot be made, but analogy with bacteria (see YEas, 1969) and rabbit reticulocytes (Gupta, 1968) favors the nonsenserole for UGA in yeast.
UAA
OCHRE
CODON
(d) Mutational
IN
93
YEAST
selectivity
Restriction of all 45 replacements to amino acids having codons that differ from UAA by one base change, despite the suitability of most, probably all, amino acids at position 2, indicates a pronounced tendency for all eight mutagenic treatments and spontaneous events to produoe single base pair substitutions rather than multiple base substitutions. This tendency also characterizes induced and spontaneous substitution mutation in E. wli (Yanofsky, Ito & Horn, 1966; Weigert, Gallucci, Lanka & Garen, 1966) and spontaneous substitution mutation in man (Vogel, 1969). This restriction of the replacements also supports the equality of the genetic code in yeast and bacteria, with respect to the amino-acid codons that differ from UAA by one base, and permits assignment of codons for position 2 in the revertants. The nine triplets that differ from UAA by single base changes are listed in the second column of Table 3, just after the amino acids they encode. UAG (Stewart t Sherman, manuscript in preparation) and possibly UGA are nonsense codons in yeast. As shown in Table 2, at least six and possibly all seven of the remaining triplets are efficiently translated as amino-acid codons. The nine DNA substitutions that generate these nine triplets are listed in Table 3, to the right of the triplets and the amino acids they encode. A 1: 1 correspondence connects the DNA substitutions with the amino-acid replacements, except for the two transversions at the third base position, which both yield a tyrosine codon, and the transitions at the second and third base positions, which generate nonsense. The relative frequencies of the six amino-acid replacements give directly the relative frequencies of the corresponding base-pair changes. Besides failing to reveal A-T -+ G*C! at the second and third bases of UAA, the replacements do not register frameshift mutations or changes from G *C. Within these limitations, spontaneous events, X-ray irradiation and the combined alkylating agents ethyl methanesulfonate, diethylsulfate and methyl methanesulfonate exhibit no selectivity with respect to the base position affected nor with respect to the three kinds of substitution from A-T pairs. Since ethyl methanesulfonate exhibits a high selectivity for G*C -+ A-T in reversion of the initiation mutants of yeast (Stewart et al., 1971), the apparently random mutagenic effect of the alkylating agents on A-T pairs in cyl-9 may underlie substantial hidden effects on G-C pairs. Nitrous acid primarily causes the only detectable transition, A-T --t G*C at the first base, and ultraviolet irradiation causes only this transition. However, these selectivities are not exhibited during reversion of the nonsense triplet UAG that encodes residue position 9 in cyl-179 (Stewart t Sherman, manuscript in preparation). In that case, revertants indicate that ultraviolet irradiation and nitrous acid, acting to revert cyl-179, cause A*T -+ T-A transversions nearly as frequently as A-T -+ G*C transitions. The substitutional mutagenic speoificities of ultraviolet light and nitrous acid on A-T pairs depend on their location in the cyl gene. Additional replacement data in revertants of several cyl nonsense mutants is being accumulated, which extends these observations on mutagenio specificity in yeast. Preliminary reports have previously suggested that the specifioity of ultraviolet mutagenesis is affected by the position of the nonsense codon within the cyl gene (Sherman, Stewart, Cravens, Thomas t Shipman, 1969) and by the presence of genes conferring sensitivity to ultraviolet light (Lawrence, Stewart, Sherman & Thomas, 1970). (e) Luck of reinitiation Since termination
beyond the oyl-9 site
codons can activate nearby, potential
translation
initiation
sites
t Abbreviations used: EMS, N-methyl-N’-nitro-N-nitrosoguanidine;
UAB UAU UAC
None Tyrosiue Tyrosine
AAA aAA
CAA
UGA UUA UCA
acid
2
Codon
None Leucine Serine
Glutamine Lysiue Glutamic
Amino acid rePl=ing glutamic acid
ethyl
methauesulfonate; NA, nitrous
Third Third Third
Second Second Second
First First First
BeseinUAA affected by reversion
acid.
DES,
Total diethylsulfate;
MMS,
methyl
0 4
2 6
2 6
I
-
-
_
_ 1 0
A.T-,G.C A-T-T-A A.T+C-G
1 1
2 1 0
EMS
-
-
1 1 0
2 0 1
0 1
X-ray
None
A.Tm,G.C A*T+T.A A.T-&*G
A-T-t GC A.T-+T.A A.T+C!.G
Base change of reversion
0 1
2 3
_
_ 1 0
0 0 0
Mutagent MMS
methauesulfonate;
0 3
-
-
1 1 0
DES
Frequencies of amino-acid replacements in i.so-1-cytochromes c from cyl-9 the wrrespmding base-pair changes
TABLE 3
-
NIL,
0 0
3
4
-
11
5
-
0 0
0
-
11 0 0
U.V.
0
_ 1 0
0
0
4
NA
l-nitresoimidezolidone-2;
0
_
-
0
0
0 0
1
1
-
2
1
NC
2
NIL
revertants and
7
4 3
NG,
45
-
-
1
6
24
Total
UAA
OCHRE
CODON
IN
YEAST
96
within genesof bacteriophage T4 (Sarabhai & Brenner, 1967) and E. co%(Grodzioker & Zipser, 1968), it is of interest to note whether the nonsensetriplet encoding position 2 alters the expression of a potential initiation site encoding position 4 of iso-l-cytochrome c. The AAG triplet encoding lysine 4 is mutated to AUG in somerevertants of cyl mutants that lack the normal AUG initiation codon; this reversion mutation permits the initiation of translation, at position 4, of one-half the normal amount of a short form of iso-1-cytochrome c that lacks the four normal NH,-terminal residues (Stewart et al., 1971). It seemslikely that the A-T + T-A transversion creating AUG at position 4 should occur at a detectable frequency, since similar A-T--t T*A transversions at position 2 resulted in six lysine and four leucine replacements in the 45 revertants described in this paper. However, the half-normal level of cytochrome c characteristic of the short-form initiation mutants was not observed during lowtemperature spectroscopic examinations of more than 309 revertants of ~~1-9, which were obtained principally by treatments with X-rays and the alkylating chemicals ethyl methanesulfonate, methyl methanesulfonate and diethylsulfate. This negative result suggeststhat the amount of the short form in cyl-9 revertants is either increased to approximately the amount characteristic of normal cytochrome c or is decreasedto significantly lessthan the characteristic one-half normal value. A choice between these alternatives, in favor of a decreasedinitiation e%oiency at the new site, is permitted by the failure to detect the short protein in the 45 revertants documented here, or in an additional 86 revertants of cyl-9 from which iso-1-cytochrome c has been extracted and analyzed by amino-acid analysis and peptide mapping (Sherman, Stewart & Lawrence, unpublished data). The extent of this reduction in e&iency of initiation is unknown; possibly reinitiation is blocked altogether. It is uncertain whether the inability to reinitiate past a nonsensecodon is a general property in yeast, or whether a restriction is imposed on the potential initiation site encoding position 4 by the specific location of the nonsensecodon in cyl-9 or by the presence of the nearby normal initiator codon. We are indebted to Mrs E. Risen and Mrs N. Brockman for their skillful performance of some of the analyses. This work was supported in part by U. S. Public Health Service research grant GM12702 from the National Institutes of Health and in part by the United States Atomic Energy Commission at the University of Rochester Atomic Energy Project, Rochester, N.Y. and has been assigned U.S. Atomic Energy Commission Report no. UR-3490-35.
REFERENCES Barnett, L., Brenner, S., Crick, F. H. C., Shuhnan, R. G. & Watts-Tobin, R. J. (1967). Phil. Trans. Roy. Sot. London, Ser. B252, 487. Beaudet, A. L. & Caskey, C. T. (1971). Proc. Nat. Ad. Sci., Wash. 68,619. Benzer, S. & Champe, S. P. (1962). Proc. Nat. Acud. Sk., Waeh. 48, 1114. Brenner, S., Barnett, L., Katz, E. R. & Crick, F. H. C. (1967). Nature, 213, 449. Brenner, S. & Beckwith, J. R. (1965). J. Mol. Biol. 13, 629. Brenner, S. & Stretton, A. 0. W. (1965). J. Mol. Biol. 13, 944. Caskey, C. T., Beaudet, A. & Nirenberg, M. (1968). J. Mol. Biol. 37, 99. Caskey, C. T., Tompkins, R., Scolnick, E., Caryk, T. & Nirenberg, M. (1968). Science, 162, 135. Champe, S. P. & Benzer, S. (1962). J. Mol. Biol. 4, 288. Gilmore, R. A., Stewart, J. W. & Sherman, F. (1971). J. Mol. Biol. 61, 157. Grodzicker, T. & Zipser, D. (1968). J. Mol. Biol. 38, 305. Gupta, N. K. (1968). J. Biol. Chem. 243, 4969.
96
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ET
AL.
Lawrence, C. W., Stewart, J. W., Sherman, F. & Thomas, F. L. X. (1970). Geenet&, 64, 036. Lu, P. & Rich, A. (1971). J. Mol. Bid. 58, 613. Marshall, R. E., C&key, C. T. & Nirenberg, M. (1967). Science, 155, 820. Nerita, K. & Titani, K. (1969). J. Bhchmn. Tokyo, 65, 259. Nichols, J. L. (1970). Nature, 225, 147. Parker, J. H. & Sherman, F. (1969). Genetim, 62, 9. Sarabhai, A. & Brenner, S. (1967). J. Mol. Bid. 27, 146. Sarabhai, A. S., Stretton, A. 0. W., Brenner, S. & Bolle, A. (1964). Nature, 201, 13. Sherman, F. & Stewart, J. W. (1971). Ann. Rev. Tenet. 5, 267. Sherman, F., Stewart, J. W., Cravens, M., Thomas, F. L. X. & Shipman, N. (1969). Genetic+ 61, s&i Sherman, F., Stewart, J. W., Parker, J. H., Inhaber, E., Shipman, N. A., Putterman, G. J., Gardieky, R. L. & Margoliaeh, E. (1968). J. BioZ. C&m. 248, 6446. Sherman, F., Taber, H. & Campbell, W. (1966). J. Mol. B&Z. 13, 21. Sdl, D., Cherayil, J. D. & Bock, R. M. (1967). J. MoZ. BioZ. 29, 97. Stewart, J. W., Sherman, F., Shipman, N. & Jackson, M. (1971). J. BioZ. Chem. 246, 7429. Stretton, A. 0. W. & Brenner, S. (1966). J. MoZ. B&Z. 12, 466. Vogel, F. (1969). Humqenetik, 8, 1. Weigert, M. G., Gdlucci, E., Lanka, E. & Garen, A. (1966). Cold Spr. Had. Syv. Qwmt. Biol. 11, 145. Weigert, M. G. t Garen, A. (1966). Nature, 206, 992. Weigert, M. G., Lanka, E. L Garen, A. (1967). J. Mol. BioZ. 28, 391. Yanofsky, C., Ito, J. & Horn, V. (1966). Cold Spr. Harb. Symp. Quant. BioZ. 31, 151. YEas, M. (1969). The Biological Code, p. 140. New York: American Eleevier.
(1972) 68, 8346
Demonstration of the UAA Ochre Codon in Bakers’ Yeast by Amino-acid Replacements in Iso-l-cytochrome c JOHN W. STEWART,FBED FRED L.X.THolldas
SHEBMAN,~XARYJA~KSON ANDNANCYSHIPMAN
of Radiation Biology and Biophysics University of RochesterSchool of Medicine and Dentistry Rocheder, New York 14642, U.S.A. Department
(Received13 December1971) Iso-1-cytochromes c from 45 intragenic rev&ants of the cyl-9 mutant of bakers’ yeast carry at position 2 one of the following six amino acids: glutamic acid, glutamine, serine, tyrosine, leucine and lysine. The replacement amino acids and the genetic code identify the cyl-9 lesion ae a base-pair substitution creating UAA at the messenger RNA template for position 2. Since, in addition, the cyl-9 mutant lacks iso-1-cyt~chrome c but can be suppressed by super-suppressor genes, this UAA triplet is a nonsense codon in yeast. The absence of other amino acids among the replacements suggests that UGA also is a nonsense codon in yeast. When acting to revert the ~~1-9 U&4 nonsense lesion, X-rays, some alkylating chemicals and spontaneous events cause substitutions of A*T base pairs with no apparent selectivity, whereas ultraviolet irradiation and nitrous acid selectively induce A*T-+ G-C. Single substitutions account for all 45 reversions either obtained spontaneously or induced by eight different mutagenic treatments. Reinitiation past the nonsense codon at position 2 fails to occur at a detectable level, although an efficient translation initiation site is accessible by mutation of the AAG, lysine 4 codon to AUG, in other mutant strains that lack the normal initiation codon.
1. Introduction Nonsensecodons were l3rst recognized in certain rII B mutants of bacteriophage T4 (Benzer & Champe, 1962 ; Champe & Benzer, 1962). Because they could be reverted by a base analog, they were believed) to be base-substitution mutants. Since they were suppressed in certain host mutants, they were believed to generate defects that could be repaired during translation of messengerRNA. The sites occupied by the mutants couldcontrol unessential regions of a protein, since some were located in a region of the B cistron that could be bounded by certain frameshift mutations or be deleted sltogether without deleterious effect, and since others fell in the A cistron, but were expressed as rI.l B mutants when the two cistrons were fused by deletions. Only if these mutations had generated coding units that did not correspond to any amino acid could these circumstances be readily understood. The different pattern of suppressionof nonsensemutations led to the distinction of amber and ochre mutants (Brenner & Beckwith, 1965). Amino-acid replacements in revertants of ember mutants of the structural genefor alkaline phosphatesein Escherichiu coli were found restricted to those having codonsdiffering by one basefrom UAG, 83
84
J. W. STEWART
ET
AL.
and this coincidence was understood to imply that UAG is the non-coding, amber triplet (Weigert & Garen, 1965). In this way also, the ochre nonsensecodon was identified as UAA (Weigert, Lanka, & Garen, 1967). A third suppressible nonsense triplet has been assignedthe identity UGA by the use of mutagens having known modes of action (Brenner, Barnett, Katz t Crick, 1967). Bacterial suppressors specific for UGA, UAG, or both UAG and UAA, suppressedall 14 different nontemperature-sensitive, base analog mutants detected in comprehensive mutational studies of the expendable region of the rII B cistron, and they also suppressedsix of the seven carefully analyzed barriers to reading frameshifts in this region (Barnett, Brenner, Crick, Sohulman & Watts-Tobin, 1967). Furthermore, only these three triplets remain unassigned as amino-acid codons by in vitro studies of the coding properties of all 64 nucleotide triplets (reviewed by YEas, 1969). UAA, UAG and UGA therefore are evidently the only nonsensecodonsin E. coli. Mutational introduction of the amber triplet into the normal reading frame (Brenner & Stretton, 1965) brings about the termination of growth of nascent polypeptide chains, at the residue coded by the triplet just preceding UAG (Stretton & Brenner, 1965), and causestheir release(Sarabhai, Stretton, Brenner & Bolle, 1964). The ochre codon UAA occupies the site expected for a natural chain termination codon in the coat protein cistron of bacteriophage R17 (Nichols, 1970). All three nonsensecodons signal the release of growing pepticles from peptidyl-tRNA on cell-free ribosomesof E. coli (Caskey, Tompkins, Scolnick, Caryk & Nirenberg, 1968). The three nonsense triplets, individually or in tandem combination (Nichols, 1970; Lu & Rich, 1971), are apparently the normal signals for chain termination in E. di. Studies with eucaryotic cell-free preparations have left UAA and UAG among the eight triplets not yet assignedas amino-acid codons (Marshall, Caskey t Nirenberg, 1967; S6ll, Cherayil & Bock, 1967; Caskey, Beauclet & Nirenberg, 1968; Gupta, 1968), and suggestthat the three bacterial nonsensetriplets can signal the releaseof pepticles from peptidyl-tRNA in mammalian cells (Beaudet & Caskey, 1971). It would be reassuring to find that these three triplets are in fact nonsense codons in living eucaryotic cells. In this paper we show that UAA is a nonsensecodon in bakers’ yeast. Amino-acid substitutions in iso-1-cytochrome c from revertants of the cyl-9 mutant identify its lesion as UAA replacing the GAA triplet that normally encodesglutamic actid2 in the protein. The cyl-9 mutation prevents the occurrence of the protein, although residue 2 may be varied greatly or deleted altogether without adverse effect. Completing the parallel with nonsensemutants in E. co&, the cyl-9 mutant is suppressible(Gilmore, Stewart & Sherman, 1971).
2. Materials and Methods (a) Genetie nmmlatolre
and strain.9
The symbol CYl denotes the wild-type structural gene for iso-1-cytoohromec; cgll denotesmutants of this gene, which are substantially deficient in amount or activity of iso-1.cytochrome c. Allele numbers following the cyl symbol, as in cyl-9, distinguish mutants derived independently. Intragenic revertants are designated by CYl followed by the allele number of the cyl parent strain, followed in turn by allele letters to distinguish independent revert&nts. For example, CYl-9-A and CYI-9-B designate two intragenic revertants of ql-9. The original cyl-9 mutant was induced by ultraviolet irradiation and was detected by the benzidine staining method (Sherman et al., 1968). The two haploid strains used in this
UAA
OCHRE
CODON
IN
85
YEAST
study, JP109-3A and JP109-6A, are segregants having the genotype OL ~~1-9 p+, which were obtained from a cross of the origi.nal cyl-9 p- strain with a normal strain (Parker & Sherman, 1969). Independent revertants were selected on k&ate medium (Sherman et al., 1968) after no treatment or after treatments of either 1.5 krad. of X-rays or 1600 ergs mmma of ultreviolet irradiation, or 15 mm with 50 mM-nitrous acid, or 30 mm with 3 ma6-l-nitrosoimidazolidone-2 (Sherman et al., 1968; Stewart, Sherman, Shipman & Jackson, 1971; Parker 6t Sherman, 1969), or 40 min with 2% (v/v) ethyl methanesulfonate. Revertants were obtained with diethylsulfate, N-methyl-N’-mtro-N-nitrosoguanidine and methyl methanesulfonate by streaking a diluted cell suspension on glycerol medium (1% Bscto-yeast extract, 2% Bacto peptone, 3% (v/v) glycerol and 2% agar), and placing a paper disc with the mutagen on the surface of the plate. The discs were removed after 2 days of incubation and the plates were replica plated onto lactate medium. Over 300 revertants were purified by subcloning and the subolones were examined for their cytochrome content by lowtemperature spectroscopy. Forty-six revertants with approximately normal amounts of cytochrome c, judged by spectroscopy of whole cells, were chosen for this study. Forty of these were analyzed genetically to determine whether the back mutation was at the cyl locus (Sherman et al., 1968). The revertants were crossed to normal haploid strains and 20 segregants from each of the sporulated diploids were examined for their cytochrome spectra. (b)
Growth
of yea&
and
preparation
of iso-1-cytochrome
c
Ten-liter batches of the cyl-9 revertants were grown as described previously (Stewart et al., 1971), yielding from 0.5 to 1.0 kg of wet cells. Iso-1-cytochrome c was extracted and purified by the methods of Sherman et al. (1968). To achieve adequate purity in a few instances, a repetition of the chromatographic purification on CG50 cation-exchange resin (200 to 400 mesh) was required. Yields of iso-1-cytochrome c were 20 to 90 mg /lO-l.batch. (c) Identijicution
of structural
changes
in iso-1-cytochrome
c
enzymic digestion, peptide mapping, The methods used for amino-acid analysis, sequential Edman degradation and chromatographic identification of the phenylthiohydantoins have been described (Stewart et al., 1971). Proteins were hydrolyzed in constsntboiling hydrochloric acid in sealed, evacuated tubes at 111°C for 20 hr, and the hydrolysates were analyzed with a Beckman-Spinco amino-acid analyzer, model 120C. Molar compositions were based on a theoretical content of 79 acidic and neutral amino-acid residues, other then cystine and methionine, and 23 basic amino-acid residues per molecule of protein (Narita & Titani, 1969), except in those instances when 78 and 24 gave more acceptable results. Tryptic and chymotryptic digests of Bamples of 0.1 pmole of oytochrome c were prepared by 2.75 hr of reaction at 37°C of 1% w/v cytochrome c and 0.1% w/v enzyme in 0.25% w/v ammonium bicarbonate. The digests were lyophylized and were subjected to electrophoresis on Whatman no. 3 MM paper wet with pyridine/acetic acid/water (5:0.18: 95), pH 6.5, for 1.66 hr at 20 V/cm, and then to descending chromatography in n-butanol/pyridine/acetic acid/water (15: 10: 3 : 12) for 16 hr. The peptide maps were developed by sequential dipping through the collidine/ninhydrin reagent, the Ehrlich reagent for tryptophan, and the Pauli reagent for histidine and tyrosine, in that order. Samples of 0.3 pmole of cytocbrome c were subjected to sequential Edman degrsdation and the phenylthiohydantoins were identified by thin-layer chromatography on silica gel.
3. Results The cyl-9 mutant was recognized as possibly a nonsense mutant in the course of a program of amino-acid analysis and peptide mapping of iso-I-cytochromes c from revertants of cyl mutants which were altered in unknown ways (for example, 888 Sherman et al., 1968). Spectroscopic examinations of whole ceIIs of cyl-9 strains and growth tests on lactate medium indicated a level of total cytochrome c equal to no more than the normally small content of iso-2-cytochrome c. The amino-acid composition of iso-l-cytoohrome c from rev&ant C yl-9-C appeared unaltered, but normal
86
J. W. STEWART
ET
AL.
peptides C-l and T-l were missing from the chymotryptic and tryptic digests, respectively, and each was replaced with a more basic peptide (Sherman et al., 1963). These analytical results, when viewed in the knowledge that C-l and T-l are the NH,-terminal peptides Thr-Glu-Phe and Thr-Glu-Phe-Lys (Stewart et al., 1971), suggestedthat glutamic acid 2 may have been replaced by glutamine. Since codons for glutamic acid and glutamine are related to the ochre and amber nonsensetriplets by single basechanges(seeYEas, 1969), these observations prompted a detailed examination of the iso-1-cytochrome c content of cyl-9, and of the structural changesin iso-1-cytochromes c from many cyl-9 revertants. (6) Absenceof iso-f-cytochrome c in cyl-9 Cytochrome c was extracted from the cyl-9 mutant JPIOS-BA and was chromatographed on cation-exchange resin. The cytochrome c eluted at a single position characteristic of iso-2-cytochrome c. The total yield was approximately 2.5 mg from an amount of yeast corresponding to 0.44 kg dry wt. This yield is within the normal range of iso-2-cytochrome c concentrations, 6 to 30 mg/kg dry weight (Sherman & Stewart, 1971; Gilmore et al., 1971; Stewart et al., 1971). No other chromatographic zone of cytochrome c was observed, although a zone of iso-1-cytochrome c equal to somewhat lessthan 10 o/oof this amount of iso-2-cytochrome c would have been seen. Since the level of iso-2-cytochrome c in both normal and cyl mutant strains is usually slightly lessthan 5% of the level of iso-1-cytochrome c in normal strains (Sherman, Taber t Campbell, 1965; Gihnore et al., 1971; Sherman t Stewart, 1971; Stewart et aZ., 1971), this result fixes a conservative upper limit of iso-1-cytochrome c content in JP109-6A at 0.5% of the normal amount. (b) Iso-I-cytochromes c in revertants of cyl-9 (i) Selection of revertunts Over 300 revertants that exhibited approximately normal growth on lactate medium were selected after treatments of ~~1-9 strains with various mutagens and after no treatment. Normal growth responseindicates the presenceof more than 10% of the normal total cytochrome c activity for lactate utilization (Gilmore et al., 1971). Almost all these revertants revealed approximately normal intensities of the cytochrome a-bands. The cytochrome a-band intensities provide a sensitive assay of total cytochrome c content: for example, spectroscopic estimates of 50% normal levels in whole cells were confirmed by extraction procedures (Stewart et al., 1971). Forty revertants containing normal amounts of cytochrome c were tested genetically for closelinkage between the reversion site and the cyl-9 site. One revertant, B-929, was clearly due to s mutation at a gene distant from cyl, but the other 39 strains appeared due to intragenic reversion. The exceptional strain, B-929, yielded a normal amount of total cytochrome c, which was entirely iso-2-cytochrome c on the basisof preparative chromatographic results, peptide maps and amino-acid composition. Closerscrutiny of the low-temperature spectrum of whole cells of this strain revealed an unusually narrow c,-band that was shifted slightly towards the blue. This abnormal spectroscopic behavior might have been anticipated as an indication of iso-2cytochrome c, since low-temperature spectrophotometric tracings of purified cytochromesc show that the c,-band of iso-2cytochrome c is slightly narrower than that of iso-1-cytochrome c and is shifted O-5 nm toward lower wavelengths (Sherman 8r Stewart, 1971). In similar studies with other cyl mutants, we have found (Sherman &
UAA
OCHRE
CODON
IN
YEAST
87
Stewart, unpublished experiments) that comparably small proportions of the revertants are due to extragenic suppressorswhich elevate the content of iso-2-cytochrome c to a level normally found for iso-1-cytochrome c. In all cases, these revertants could be recognized by their narrow and shifted c,-band. On the other hand, iso-l-cytochrome c was obtained from all extracted revertants that showed the normally intense but normally broad c,-band in spectra of whole cells, and genetic tests of these strains have always indicated that they were due to an intragenic reversion event (for example, seeSherman et al., 1968; Stewart et al., 1971). These observations indicate that revertants exhibiting normal spectra, with respect to the width and exact position as well as the relative intensity of the c,-band, are in all likelihood intragenic revertants. The 39 intragenic revertants that were tested genetically, as well as the six X-ray-induced revertants that contained normal c,-bands and normal amounts of iso-1-cytochrome c, are listed in Table 1. (ii) Iso-I-cytochromes c Approximately normal yields of iso-l-cytochrome c were obtained from all 45 revertant strains in Table 1. Amino-acid compositions of iso-l-cytochromes c in the 45 revertants are listed in Table 2. Because only five different types of composition were apparent, only the averages of all compositions judged to be identical are given. The five compositional types include the normal and four abnormal ones,which differ from the normal by the loss of one residue of glutamic acid and by the gain of one residue of either lysine, serine, leucine or tyrosine per molecule of protein. To indicate the reliability of the assignmentsof the 45 individual analysesto the five groups, also listed in Table 2 are the ranges of the extreme values for each amino-acid residue for all 45 proteins, normalized by the appropriate addition of 1.00 residue of glutamic acid and subtraction of l-00 residue of the extra amino acid. The integral compositional changes of the iso-1-cytochromes c in all 45 revertants are listed in Table 1. Peptide maps of all 45 proteins are summarized in Figure 1. Six different types of tryptic and chymotryptic peptide maps were found. They distinguish the samefive sets of proteins as the compositions, but subdivide the proteins having normal compositions into a classhaving normal peptide maps and a classhaving altered peptide maps. All of the abnormal peptide maps lack spots C-l and T-l but contain all of the other normal spots, as well as extra spots, which permit the distinction of the five altered types. The missing spots C-l and T-l are the NH,-terminal chymotryptic and tryptic peptides, Thr-Glu-Phe and Thr-Glu-Phe-Lys, respectively (Stewart et al., 1971). The extra spots for all 45 proteins are listed in Table 1. The net changesin composition and in peptide maps of all 45 iso-l-cytochromcs c are grossly consistent with the replacement of glutamic acid 2 with any of six different amino acids, including glutamine and glutamic acid. The changesin the peptide maps may be examined in more detail, to test this suggestion. The new peptides in the chymotryptic digests of all 44 altered proteins, except those containing extra lysine, are electrophoretically neutral at pH 6.4, whereasthe new chymotryptic peptide from the proteins having extra lysine exhibits a mobility towards the cathode approximately equal in magnitude to the mobility of peptide C-l, Thr-Glu-Phe, towards the anode. Electrophoretically, these changesare consistent with replacement of glutamic acid 2 in Thr-Glu-Phe with neutral amino acids including glutamine, and with lysine. Chromatographically, the new chymotryptic peptides are related to Thr-Glu-Phe in
88
J.
W.
STEWART
TABLE
ET
AL.
1
Alterations of iso-1-cytochrm c in 45 inbragenicrevertants of cyl-9 straimt Rev&ant
strain
Alterations
Number
Genotype
Mutagen%
B-496 B-496 B-497 B-829 B-830 B-831 B-832 B-833 B-834 B-835 B-836 B-837 B-913 B-914 B-915 B-916 B-917 B-918 B-919 B-921 B-922 B-923 B-924 B-926 B-1038 B-1080 B-1081 B-1082 B-1089 B-1090 B-1091 B-1098 B-1099 B-1100 B-1290 B-1291 B-1959 B-1960 B-1961 B-1962 B-1963 B-1964 B-2069 B-2070 B-2071
CYl-9-A CYl-9-B GYl-9-C CYl-9-D CYl-9-E CYl-9-F CYl-9-G CYl-9-H CYl-9-I CYl-9-J CYl-9-K CYl-9-L CYl-9-M CYl-9-N CYl-9-o CYl-9-P CYl-9-Q CYl-9-R CYl-9-s CYl-9-T CYl-9-u CYl-9-v CYl-9-w CYl-9-x CYl-9-Y CYl-9-z CYl-g-AA CYl-9-AB CYl-O-AC CYl-g-AD CYl-9-AE CYl-9-AF CYl-9-AG CYl-S-AH CYl-9-AI CYl-9-AJ CYl-B-AK CYl-g-AL CYl-O-AM CYl-g-AN CYl-g-A0 CYl-9-AP CYl-9-A& CYl-g-AR CYl-O-AS
U.V. U.V. . . Gale U.V. U.V. U.V. U.V. U.V. U.V. U.V. U.V. None EMS EMS EMS EMS NIL NIL NA NA NA NA NA None DES DES DES NG NC NG MMS MMS MMS NIL NIL X-ray X-ray X-ray X-ray X-ray X-ray None None None
Change in composition$ YMidueal~OleCulS None None None None None None None None None None None None None None None -Glu, +Lys -Glu, +Leu - Glu, +Lys -Glu, +Tyr None None - Glu, +Leu None None -Glu, +Tyr - Glu, +Lys -Glu, +Ser None None None -Glu, +Lys -Glu, +Tyr - Glu, +Tyr -Glu, +Leu None -Glu, +Lys - Glu, +Ser - Glu, +Tyr - Glu, +Tyr - Glu, +Lys None - Glu, + Leu -Glu, +Ser None -Glu, +Tyr
Peptide map7 replacement of C-l and T-l b b b b b b b b b b b b b b b a e d” b b b b d a E b b 1 d i a d d b” e c None
d
Replacement of glut’amic acid 2
GinII Gln Gln Gln Gln Gln Gln Gln Gln Ghl Gln Gln Gln Gln Gln LYS Leu LYS TV Gln Gln Leu Gln Gln TF LYS SW! Gln Ghl Ghl LYS Tyr TV L&l Gln Lys Ser TF Tyr LYS Ghl Leu Ser Glu TF
t The cyl-9 strain that was revert-ad to produce revertant strains B-496, B-496 and B-497 was JP109-3A; the cyl-9 strain from which all other revertants were prepared was JP109-6A. $ The abbreviations of mutagens used are as follows: u.v., ultraviolet light; EMS, ethyl methanesulfonate; NIL, 1-nitrosoimidazolidone-2; NA, nitrous aoid; DES, diethyl sulfate; NC, iv-methylN’.nitro-N-nitrosoguanidine; MMS, methyl methanesulfonate. $ Amino-acid compositions of the proteins are in Table 2. 1 Peptide maps of chymotryptic and tryptic digests are in Fig. 1. jl Glutamine was directly shown, by chromatography of the phenylthiohydantoin of residue 2, only for CYl-9-A and CYl-9-B; in all other cases it was inferred from the neutrality, at pH 6.4, of the replacement of Thr-Glu-Phe and from the normal amino-acid composition.
1
TryptoPh
2
Type
of composition
6
1
1'7.26 3.68 3.06 11.13 7.65 4.10 8-64 4.37 II-85 7.24 1.82 3.06 1.82 3.81 7.64 4.72 3.89
-Glu,+Lys
4
1
2.26 3.06 1.74 3.79 8.74 4.73 3.90
11.90 7.18
4.01 8.48 4.39
11.18 7.61
16.15 3.86 2.99
Average
- Glu, + Ser
3
1
3.90 2.99 Il.33 7-72 4-80 8-47 4.21 11.92 7.16 2.66 3.02 l-63 3.89 7.86 4-65 3.97
16.11
residues/molecule
- Glu, +Leu
Range
16.16 3-80 3.04 11.20 7-65 4.07 9.63 4.29 Il.89 7.34 2.19 3.04 1.78 3.78 7.79 4.63 3.89
Midpoint
7xJO 1.16 2.93 1.38 3.55 7.56 4.35 3.64
11.51
16-89 3.52 2,87 10.86 7.47 3.76 9.26 3-88
of 46 normalized Minimum
16.42 4.08 3.20 11.84 7.83 4.39 9.81 4.70 12.27 7-67 3.22 3.16 2.09 4.00 8.02 4.91 4.14
values ilfaximum
Results are from single analyses of 20-hr hydrolysates of each protein in 6 N-HCl at 11 l”C, for all residues except tryptophan; values for tryptophan are based on results of staining peptide maps with an Ehrlich reagent. Molar values were calculated from mole fraotions, assuming 79 neutral and acidic residues and 23 basic residues, excluding tryptophan, oystine and methionine, per mole of normal protein; 78 and 24 were assumed instead for the - Glu, +Lys proteins. Values reported are averages of values for all proteins judged identical; the number of protein analyses averaged is shown at the bottom of each column. To obtain a single measure of dispersion of the values for all 46 proteins, the analysis of each protein was either left unaltered or adjusted to the normal by addition of I.00 residue of glutamio said and subtraction of I.00 residue of tyrosine, Iysine, leuome, or serine, as diotated by the change of its compositional type from the normal; the extremes of these 45 normalized values and the midpoints of these extremes are recorded.
Total
25
Phenylalanine
proteins
7.23 2.26 3.05 1.83 3.79 7.79 5.72 3.86 1 7
TyrOShlt3
11.96
16.20 3.79 3.01 Il.17 7.72 4.05 8.66 4.11
- Glu, f Tyr
7.22 I.98 3.04 1.85 3.86 7.89 4.74 3.92
7.66 3.94 9.48 4.28
11.10
3-77 3.02
1621
Normal
11.91
acid
acid
acid
Glycine Allmine Half-cystine Vsline Methionine Isoleuoine Leucine
PrOline
LyfJin0 Hi&dine Arginine Aspartio Threonine serine Glutamio
Amino
TABLE
Amino-acid composition of 45 iso-1-cytochromesc
90
J.
W.
STEWART
AL.
Electrophoresis
I -: Origin I
ET
Orlgln J
Chymotryptlc
:I
Origfn J
digest
Tryptic
digest
FIQ. 1. Cumulative peptide maps of 45 iso-1-cytochromes c. Outlined areas represent nermal spots on ninhydrin-stained peptide maps of iso-1-cytochrome c from wild-type bakers’ yeast and from revertant strain B-2070 (CYl-S-AR). C-l and T-l are normal NH,-terminal peptides and are missing from peptide maps of all revertant strains except B-2070. (0) Mark centers of new, ninhydrin-positive peptides of &ered proteins. Lower case letters designate amino-acid replaaemerits of glutamic acid in proteins yielding these new peptides, as follows: lysine, a; glutamine, 5; se&e, c; tyrosine, d; and leucine, e. In all 90 peptide maps, spots at normal positions gave normal color responses with the oollidine-ninhydrin, Ehrlich and Pauli reagents. All new spots except the lower of the tryptio peptides a gave yellow colors initielly with the ninhydrin reagent. All new spots gave negative Ehrlich color reactions for typtophan, and all new spots except d, which were tyrosine-positive, failed to react positively with the Peuli reagent. Left panel shows a chromatogram of amino acids, applied to a peptide map at the same height as visible heme peptides after electrophoresis but before chromatography. Numbers designate amino acids as follows: lysine, 1; glutamine, 2; glutamic acid, 3; serine, 4; tyrosine, 5; phenylalanine and leutine. 0.
much the same ways as the corresponding amino-acid replacements are related to glutamic acid, as shown in the left panel of Figure 1, with one exception. The single exception is the uppermost of the new peptides d in chymotryptic digests of proteins having extra tyrosine; it is relatively too high on the chromatogram. The yield of this upper peptide d is low, since it gives a relatively faint ninhydrin spot and is revealed primarily by its slightly positive response to the Pauli reagent ; in contrast, the lower peptide d is strongly responsive to both reagents. The upper peptide d may be Thr-Tyr, which could result from chymotryptic digestion of Thr-Tyr-Phe; the confirmatory presence of phenylalanine was not detected, but small amounts of phenylalanine would be masked by normal spots. Also, it could result from digestion of larger NH,-terminal fragments. Similar analysis of the tryptic peptide maps also permits the conclusion that neutral
TJAA
OCHRE
CODON
IN
YEAST
91
residues replace glutamic acid 2 in Thr-Glu-Phe-Lys with the exception of the apparently normal protein in CYl-9-AR and the proteins having extra Iysine. The tryptio digest8 of proteins having glutamic acid replaced by lysine contain two new peptides, a in Figure 1, which are both strongly responsive to the ninhydrin reagent. Both are more mobile toward8 the cathode than the other new peptides. These two peptides a are plausibly Thr-Lys and Phe-Lys, the expected product8 of tryptic digestion of Thr-Lys-Phe-Lys. The uppermost tryptic peptide a may be Thr-Lys, for it gave a yellow color initially during reaction with the ninhydrin reagent; in our experience yellow color8 are given by peptides only when they carry NH,-terminal residues of threonine, serine, glycine or asparagine. It is significant that all of the new spots, in both enzymic digests, except for the lower of the new tryptic peptides a from the proteins having extra lysine, gave an initial yellow color during reaction with the ninhydrin reagent. This result indicate8 a retention of threonine at the NH,-terminus of all the proteins. Retention of phenylalanine at position 3 is suggested by the chymotryptic peptide maps, to account for the universal presence of peptide C-2, which includes residue8 4-14 (Stewart et al., 1971). These detailed examinations of the peptide map8 tend to 6x the site altered as position 2. The amino-acid sequence of the three NH,-terminal residues of the iso-l-cytochromes c from the first two revertants GYl-9-A and CYl-9-B was determined by sequential Edman degradation. The NH,-terminal sequence of both proteins was Thr-Gln-Phe.
4. Discussion (a) The ~~1-9 mutation is a nonsense mutation The drop in iso-l-cytochrome c content to below 1% of the normal level in strain JP109-6A reveal8 substantial unacceptability of the cyl-9 mutational lesion. The revertability of the lesion with a variety of mutagens suggests that the mutation is due to a base-pair substitution rather than deletion or insertion of base pairs. This suggestion is supported by the restriction of the site of primary structural alterations in the 45 revertant iso-l-cytochromes c to a single residue position. Furthermore, the aminoacid replacement8 observed are consistent with single base-pair substitution in the cyl-9 mutation, as will be detailed in section (b) below. The lesion probably does not prevent the formation of mRNA, since it is weakly suppressed by class 1, set 1 supersuppressors, which may act through altered transferRNA’s (Gilmore et al., 1971). The cyl-9 mutation is apparently a substitution mutation that prevent8 translation or causes gross instability of iso-l-cytochrome c. A single amino-acid change at position 2 that causes gross abnormality of the protein seems an unlikely explanation for the deficiency in cyl-9, since the replacements in the revertants, glutamic acid, lysine, glutamine, serine, tyrosine and leucine, cover most of the range of structural diversity of amino acids. Furthermore, position 2 is excluded altogether in a short form of iso-l-cytochrome c, which lacks the normal, first four NH,-terminal residues, without serious impairment of function (Stewart et al., 1971). Nevertheless, certain residues at position 2 might act in especially deleterious ways. Only three such amino acid8 come to mind. Cysteine might interfere with heme addition to cysteinyls 19 and 22 in the apoprotein, or might react adversely with the single cysteinyl residue at position 107. However, cysteine does satisfactorily occupy the nearby homologous position 7 in a revertant strain B-2178 prepared from
92
J.
W.
STEWART
ET
AL.
~~1-183 (Stewart & Sherman, unpublished data). Methionine or histidine might provide incorrect ligands to the heme iron, but methionine satisfactorily occupies a neighboring position, at the NH,-terminus of iso-l-cytochrome c in revertants of initiator mutants (Stewart et al., 1971). If the cyl-9 mutation is in fact a single basepair change affecting the triplet encoding position 2, then it must be consideredin all likelihood a nonsensecodon. (b) The nonsense codon in cyl-9 is UAA Only one residue position appears altered in all 45 revertant proteins. Position 2, which is normally glutamic acid, has become glutamine, lysine, leucine, serine, or tyrosine, or remained glutamic acid. According to the bacterial genetic code (see Y&s, 1969), these amino acids constitute the entire set of amino acids that have codons that differ from UAA by one base change, and are all but one of the amino acids that have codonsthat differ from UAG by one basechange. They could all arise from UAPu in cyl-9 by a single base change. In contrast, instances of multiple base changeswould be required to generate codonsfor all these amino acids from any other triplets. This correlation identifies the altered messengerRNA triplet corresponding to position 2 in cyl-9 as UAPu. The cyl-9 mutation was apparently a G-C-+ T.A substitution affecting the first base of the codon for glutamic acid 2, GAPu. Complete identification of the UAPu triplet depends upon negative information. Tryptophan is not among the 45 amino-acid replacements at position 2. The UGG tryptophan codon in yeast (Stewart & Sherman, manuscript in preparation), may be derived from UAG by an A-T --+ G*C transition at the second base position. This change probably occurred at a detectable frequency, since the A-T -+ G*C transition at the iirst base,which generated the CAPu codonsfor the 24 glutamine replacements, was the preponderant changedetected, and occurred spontaneously and with seven of the eight mutagens; and since, in addition, the second baseof UAPu appearsto be as mutable as the first and third bases,judged by the frequencies of transversions (see Table 3, in section (d)). The structural variations observed at and near position 2 leave little doubt that tryptophan would be acceptable at position 2 ; substantial reduction in function would be required to preclude selection, since a 90% decreasein iso-lcytochrome c content affects only slightly the growth on lactate medium (Gilmore et al., 1971). For these reasons, the absence of tryptophan from the 45 replacements suggeststhat the UAPu triplet in cyl-9 is not UAG. Supporting this conclusion is the observation that an efficient, tryosine-inserting super-suppressorof the UAG nonsense triplet in cyl-179 (Sherman, Liebmen, Stewart dzJackson, manuscript in preparation) fails to suppress cyl-9, although other less efficient super-suppressorsthat insert tyrosine do suppresscyl-9 (Gilmore et al., 1971). By difference, the unacceptable UAPu lesion in cyl-9 is UAA. Just asin E. coti, UAA is a nonsensecodon in yeast. (c) Coding by the triplet UGA Reasonsheve been given for believing that any amino acid should be acceptable at position 2, and that the transition A*T -+ G-C affecting the secondbase of UAA in cyl-9 would have been registered among the 45 revertants if it could produce an acceptable amino acid codon. For those reasons,the UGA triplet resulting from this transition must be either a new codon for one of the amino-acid replacements, glutamic acid, lysine, glutamine, leucine, serine or tyrosine; or it must be a nonsenseoodon. A definitive choice cannot be made, but analogy with bacteria (see YEas, 1969) and rabbit reticulocytes (Gupta, 1968) favors the nonsenserole for UGA in yeast.
UAA
OCHRE
CODON
(d) Mutational
IN
93
YEAST
selectivity
Restriction of all 45 replacements to amino acids having codons that differ from UAA by one base change, despite the suitability of most, probably all, amino acids at position 2, indicates a pronounced tendency for all eight mutagenic treatments and spontaneous events to produoe single base pair substitutions rather than multiple base substitutions. This tendency also characterizes induced and spontaneous substitution mutation in E. wli (Yanofsky, Ito & Horn, 1966; Weigert, Gallucci, Lanka & Garen, 1966) and spontaneous substitution mutation in man (Vogel, 1969). This restriction of the replacements also supports the equality of the genetic code in yeast and bacteria, with respect to the amino-acid codons that differ from UAA by one base, and permits assignment of codons for position 2 in the revertants. The nine triplets that differ from UAA by single base changes are listed in the second column of Table 3, just after the amino acids they encode. UAG (Stewart t Sherman, manuscript in preparation) and possibly UGA are nonsense codons in yeast. As shown in Table 2, at least six and possibly all seven of the remaining triplets are efficiently translated as amino-acid codons. The nine DNA substitutions that generate these nine triplets are listed in Table 3, to the right of the triplets and the amino acids they encode. A 1: 1 correspondence connects the DNA substitutions with the amino-acid replacements, except for the two transversions at the third base position, which both yield a tyrosine codon, and the transitions at the second and third base positions, which generate nonsense. The relative frequencies of the six amino-acid replacements give directly the relative frequencies of the corresponding base-pair changes. Besides failing to reveal A-T -+ G*C! at the second and third bases of UAA, the replacements do not register frameshift mutations or changes from G *C. Within these limitations, spontaneous events, X-ray irradiation and the combined alkylating agents ethyl methanesulfonate, diethylsulfate and methyl methanesulfonate exhibit no selectivity with respect to the base position affected nor with respect to the three kinds of substitution from A-T pairs. Since ethyl methanesulfonate exhibits a high selectivity for G*C -+ A-T in reversion of the initiation mutants of yeast (Stewart et al., 1971), the apparently random mutagenic effect of the alkylating agents on A-T pairs in cyl-9 may underlie substantial hidden effects on G-C pairs. Nitrous acid primarily causes the only detectable transition, A-T --t G*C at the first base, and ultraviolet irradiation causes only this transition. However, these selectivities are not exhibited during reversion of the nonsense triplet UAG that encodes residue position 9 in cyl-179 (Stewart t Sherman, manuscript in preparation). In that case, revertants indicate that ultraviolet irradiation and nitrous acid, acting to revert cyl-179, cause A*T -+ T-A transversions nearly as frequently as A-T -+ G*C transitions. The substitutional mutagenic speoificities of ultraviolet light and nitrous acid on A-T pairs depend on their location in the cyl gene. Additional replacement data in revertants of several cyl nonsense mutants is being accumulated, which extends these observations on mutagenio specificity in yeast. Preliminary reports have previously suggested that the specifioity of ultraviolet mutagenesis is affected by the position of the nonsense codon within the cyl gene (Sherman, Stewart, Cravens, Thomas t Shipman, 1969) and by the presence of genes conferring sensitivity to ultraviolet light (Lawrence, Stewart, Sherman & Thomas, 1970). (e) Luck of reinitiation Since termination
beyond the oyl-9 site
codons can activate nearby, potential
translation
initiation
sites
t Abbreviations used: EMS, N-methyl-N’-nitro-N-nitrosoguanidine;
UAB UAU UAC
None Tyrosiue Tyrosine
AAA aAA
CAA
UGA UUA UCA
acid
2
Codon
None Leucine Serine
Glutamine Lysiue Glutamic
Amino acid rePl=ing glutamic acid
ethyl
methauesulfonate; NA, nitrous
Third Third Third
Second Second Second
First First First
BeseinUAA affected by reversion
acid.
DES,
Total diethylsulfate;
MMS,
methyl
0 4
2 6
2 6
I
-
-
_
_ 1 0
A.T-,G.C A-T-T-A A.T+C-G
1 1
2 1 0
EMS
-
-
1 1 0
2 0 1
0 1
X-ray
None
A.Tm,G.C A*T+T.A A.T-&*G
A-T-t GC A.T-+T.A A.T+C!.G
Base change of reversion
0 1
2 3
_
_ 1 0
0 0 0
Mutagent MMS
methauesulfonate;
0 3
-
-
1 1 0
DES
Frequencies of amino-acid replacements in i.so-1-cytochromes c from cyl-9 the wrrespmding base-pair changes
TABLE 3
-
NIL,
0 0
3
4
-
11
5
-
0 0
0
-
11 0 0
U.V.
0
_ 1 0
0
0
4
NA
l-nitresoimidezolidone-2;
0
_
-
0
0
0 0
1
1
-
2
1
NC
2
NIL
revertants and
7
4 3
NG,
45
-
-
1
6
24
Total
UAA
OCHRE
CODON
IN
YEAST
96
within genesof bacteriophage T4 (Sarabhai & Brenner, 1967) and E. co%(Grodzioker & Zipser, 1968), it is of interest to note whether the nonsensetriplet encoding position 2 alters the expression of a potential initiation site encoding position 4 of iso-l-cytochrome c. The AAG triplet encoding lysine 4 is mutated to AUG in somerevertants of cyl mutants that lack the normal AUG initiation codon; this reversion mutation permits the initiation of translation, at position 4, of one-half the normal amount of a short form of iso-1-cytochrome c that lacks the four normal NH,-terminal residues (Stewart et al., 1971). It seemslikely that the A-T + T-A transversion creating AUG at position 4 should occur at a detectable frequency, since similar A-T--t T*A transversions at position 2 resulted in six lysine and four leucine replacements in the 45 revertants described in this paper. However, the half-normal level of cytochrome c characteristic of the short-form initiation mutants was not observed during lowtemperature spectroscopic examinations of more than 309 revertants of ~~1-9, which were obtained principally by treatments with X-rays and the alkylating chemicals ethyl methanesulfonate, methyl methanesulfonate and diethylsulfate. This negative result suggeststhat the amount of the short form in cyl-9 revertants is either increased to approximately the amount characteristic of normal cytochrome c or is decreasedto significantly lessthan the characteristic one-half normal value. A choice between these alternatives, in favor of a decreasedinitiation e%oiency at the new site, is permitted by the failure to detect the short protein in the 45 revertants documented here, or in an additional 86 revertants of cyl-9 from which iso-1-cytochrome c has been extracted and analyzed by amino-acid analysis and peptide mapping (Sherman, Stewart & Lawrence, unpublished data). The extent of this reduction in e&iency of initiation is unknown; possibly reinitiation is blocked altogether. It is uncertain whether the inability to reinitiate past a nonsensecodon is a general property in yeast, or whether a restriction is imposed on the potential initiation site encoding position 4 by the specific location of the nonsensecodon in cyl-9 or by the presence of the nearby normal initiator codon. We are indebted to Mrs E. Risen and Mrs N. Brockman for their skillful performance of some of the analyses. This work was supported in part by U. S. Public Health Service research grant GM12702 from the National Institutes of Health and in part by the United States Atomic Energy Commission at the University of Rochester Atomic Energy Project, Rochester, N.Y. and has been assigned U.S. Atomic Energy Commission Report no. UR-3490-35.
REFERENCES Barnett, L., Brenner, S., Crick, F. H. C., Shuhnan, R. G. & Watts-Tobin, R. J. (1967). Phil. Trans. Roy. Sot. London, Ser. B252, 487. Beaudet, A. L. & Caskey, C. T. (1971). Proc. Nat. Ad. Sci., Wash. 68,619. Benzer, S. & Champe, S. P. (1962). Proc. Nat. Acud. Sk., Waeh. 48, 1114. Brenner, S., Barnett, L., Katz, E. R. & Crick, F. H. C. (1967). Nature, 213, 449. Brenner, S. & Beckwith, J. R. (1965). J. Mol. Biol. 13, 629. Brenner, S. & Stretton, A. 0. W. (1965). J. Mol. Biol. 13, 944. Caskey, C. T., Beaudet, A. & Nirenberg, M. (1968). J. Mol. Biol. 37, 99. Caskey, C. T., Tompkins, R., Scolnick, E., Caryk, T. & Nirenberg, M. (1968). Science, 162, 135. Champe, S. P. & Benzer, S. (1962). J. Mol. Biol. 4, 288. Gilmore, R. A., Stewart, J. W. & Sherman, F. (1971). J. Mol. Biol. 61, 157. Grodzicker, T. & Zipser, D. (1968). J. Mol. Biol. 38, 305. Gupta, N. K. (1968). J. Biol. Chem. 243, 4969.
96
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