Serine substitutions caused by an ochre suppressor in yeast

J. Nol. Biol. (1975) 94, 595-610

Serine Substitutions Caused by an Ochre Suppressor in Yeast SUSANW.LIEBMAN, JOHN W.STEWARTANDFRED SHERMAN Department of Radiation Biology and Biophysics of Rochehester School of Medicine and Dentistry Rochester, N.Y. 14642, U.S.A.

University

(Received 9 December 1974) The suppressor SUQ5 in yeast can cause the production of approximately 10 to 20% of the normal amount of iso-l-cytochrome c when coupled to the ochre (UAA) mutants cycl-2 and ~~~1-72. The iso-1-cytochromes c contain residues of serine at positions that correspond to the sites of the ochre codons. SUQ5 is efficient only in strains having the non-Mendelian factor $+, although the low amount of suppressed iso-1-oytochrome c from a $- SUQS cycl-72 strain was also shown to contain serine at the ochre site. Thus SUQS differs from the eight other characterized suppressors of UAA in yeast, which were previously shown to insert residues of tyrosine at ochre sites (Gilmore et al., 1971) and which are only effective in strains having the non-Mendelian factor $I-, since they generally cause inviability in the $+ state. Like the tyrosine-inserting suppressors, SUQS can also act on another ochre allele cycl-9, but with a very low efficiency of approximately 0.4%, while it does not appear to act at all on amber (UAG) mutants. SUQ5 was found to be 6.4 CM (centiMorgans) from tyr7 on chromosome XVI. It is suggested that the gene product of SUQ5 is serine tRNA.

1. Introduction In an extensive search for nonsense suppressors in yeast, Gilmore (1967) uncovered mutants of eight distinct loci (the class I, set 1 suppressors) that would efficiently suppress all of a set of five nutritional markers that were suspected to be nonsense mutations. Suppressors at all eight loci caused the insertion of residues of tyrosine in iso-1-cytochrome c at positions which correspond to known ochre sites in cycl mutants; in contrast, these suppressors did not appear to act on amber mutants (Gilmore et al., 1971). Suppressors that acted on some but not all of the five nutritional markers also did not act efficiently on the cycl-2 ochre allele (Gilmore & Sherman, unpublished results; see Gilmore et al., 1971). These observations, together with the fact that many amino acid replacements at the position corresponding to the cycl-2 ochre triplet are compatible with biosynthesis and function of iso-1-cytochrome c (Stewart & Sherman, unpublished results; see Sherman & Stewart, 1974), suggested that only tyrosine-inserting suppressors could act efficiently on ochre mutants. In a separate study, Cox (1965) isolated another ochre suppressor, XUQS, which can also act on the five nutritional markers used in Gilmore’s (1967) search (Liebman, 1973). However, the action of the XUQS suppressor on these markers can only be observed when it is coupled with the non-Mendelian genetic determinant called 4’. This #+ factor increases the efficiencies of several ochre suppressors (Cox, 19651971; 595

596

S. W.

LIEBMAN,

J. W.

STEWART

AND

F. SHERMAN

Liebman, 1973; Liebman et al., unpublished results). Only #- strains were used by Gilmore (1967) for the isolation of the tyrosine-inserting suppressors mentioned above. Indeed, the efficiency of Gilmore’s suppressors in #+ strains is so high that the cells generally cannot survive. In contrast, SUQS acts with such a low ehkiency in #strains that suppression is barely detectable (Cox, 1965,197l; Liebman, 1973; Liebman et al., unpublished results). However, in zj + strains, X lJQ5 can efficiently suppress nutritional ochre alleles (Cox, 1965; Liebman, 1973) as well as cycl ochre mutations. In this paper XUQ5 is shown to cause the insertion of serine at the sites of the ochre codons in the cycl-2 and cycl-7.2 mutants. In addition, we have measured the efficiencies of suppression of various cycl nonsense alleles by I,P SUQ5 and determined the position of XUQ5 on the yeast genetic map. A preliminary account of some of these results has been reported (Liebman et ab., 1974).

2. Materials and Methods Four characterized nonsense mutants, oyol-2, oyol-9, oyol-72 and oyol-179 were used in this study. The oyol-2 mutant was induced with nitrous acid and was isolated by the spectroscopic scanning procedure (Sherman, 1964), the oyol-9 mutant was induced by U.V. irradiation and was isolated by the benzidine procedure (Sherman et al., 1968) and the 0~01-72 and oyol-179 mutants were each induced with ultraviolet light and were isolated by the chlorolactate procedure (Sherman et al., 1974). The nonsense codons and the corresponding residue positions were determined for these oyol mutants by amino acid replacements in revertant iso-1-cytochromes o. The oyol-2, 0~01-72 (Stewart & Sherman, unpublished results; see Sherman & Stewart, 1974) and oyol-9 (Stewart et al., 1972) mutants have ochre codons corresponding, respectively, to ammo acid residue positions 21, 66 and 2, and the oyoI-179 mutant (Stewart & Sherman, 1972) has an amber eodon corresponding to amino acid residue position 9. The original strains containing the nonsense mutants oyol-2, oyol-9 and oyol-179 were each shown to oontain the $- non-Mend&an determinant. This was established by the isolation of viable haploid strains bearing, respectively, each of the cyol mutants as well as one of the class I, set 1 suppressors. If the strains were #+, the presence of such a suppressor would cause lethality (Cox, 1971). The #- oyol alleles were easily coupled with the $+ factor by crossing them to 4’ strains from which all progeny are $+ (Cox, 1965, 1971). Although 0~01-72 was isolated from the same parent strain, D311-3A, as were the #mutants oyol-9 and oyol-179 (Sherman et aE., 1974), surprisingly the original strain containing the allele oyol-72 was found to harbor the $+ factor. Apparently a mutation of IJ- to #+ arose during the isolation of the 0~01-72 mutant. This was established by the lethal effect of crossing the oyol-72 mutant to class I, set 1 suppressors. Furthermore, crosses of the putative #+ oyol-72 mutant with z/- SUQ5 strains lead to the increased efficiency of the SUQ5 suppressor. SUQ5, which was kindly supplied to us in 4’ and #strains by Dr B. S. Cox (University of Oxford), is an efficient ochre suppressor only in strains having the non-Mendelian determinant # +. In $- strains the action of SUQ5 is barely detectable (Cox, 1965; Liebman, 1973). It was impossible to obtain a $- oyoI-72 strain by conventional crosses and dissection since all progeny arising from diploids constructed from one or more #+ strains are # +. However, the procedure described below permitted the successful selection of such a mutant strain. When first isolated the Mendelian gene R16, kindly supplied by Dr S. B. Cox, facilitated the rapid conversion of $+ to $(Young & Cox, 1971). However, the R16 allele we obtained had lost its potency. Therefore, a $- oycl-72 strain was obtained by selecting for canavanine resistant segregants from a sporulated mating mixture of the following cross: a #+ SUQ5 oyol-72 trp2 ade5 x cc#SUQ5 CYCl R16 ade2-1 1~~1-1 oanl-100. Such resistant segregants must either have lost SUQS, become $- so that the action of SUQ5 on canl-100 was lost, or gained a new canavanine resistance marker which is not suppressed by $’ SUQ5. A resistant segregant,

OCHRE

SUPPRESSOR

SERINE

SUBSTITUTIONS

597

L-375, in which the cycl-72 allele was judged present and not suppressed by spectroscopic examination, was shown to retain SUQ5 by a cross to a #’ strain which increased the efficiency of the suppressor’s action on cycl-72. Further verification that L-375 is a $SUQS cycl-72 strain was obtained by crossing it with a $- strain and examining the phenotype of SUQ5 in the segregants. Neither the ochre nutritional markers nor cycl-72 were suppressed in any of the progeny. However, the suppressing action of SUQ5 was detected when the segregants were crossed to #+ strains. Also, a non-suppressor bearing cycl-72 segregant from this pedigree was verified to be IJ- since a cross of it with a class I, set 1 suppressor produced viable suppressor-bearing segregants. The nonsense suppressor SUPY-1 (formerly called SlJP7), is one of the class I, set 1 suppressors and inserts tyrosine at ochre sites but does not act on amber cycl mutants (Gilmore et al., 1971). The hi~is5-2 and camIalleles used in this study are assumed to be ochre since they are acted on by ochre (e.g. the I/- class I, set 1 and 4’ SUQ5) but not by amber suppressors (Gilmore, 1967; Hawthorne, 1969a,b). The tyr7-l allele, which is acted on by amber but not by oohre suppressors, is presumed to contain an amber lesion (Wawthorne, 1969a,b; Sherman et al., 1973). The Zeal-12 allele is not suppressible. (b) Genetic methods Appropriate strains were constructed by conventional procedures of crossing, sporulation and dissection. Nutritional markers were scored by growth on synthetic glucose medium containing 0.67% Baoto-yeast nitrogen base (without amino acids), 2% glucose, 1.5% Ionagar (Wilson Diagnostics, Inc.), and appropriate amino acids. The segregation of the and suppression SUQ5 gene was scored by suppression of the h&5-2 allele. The segregation of the cyclcl genes was scored from the levels of cytochrome c, estimated by spectroscopic examinations of whole cells at low temperature ( - 190°C) (Sherman & Slonimski, 1964). For several representative strains, low-temperature speotrophotometric recordings were also made using a modified Cary model 14 spectrophotometer (Sherman et al., 1968). (c) Quantitative

determination

of cytochrome c

A determination of total cytochrome c content was made by spectrophotometrio measurement of quantitatively extracted cytoohrome c from known amounts of yeast amounts of the iso-l- and iso-2-oytoohromes c were (Sherman et al., 1965). The relative determined speotrophotometrically after ohromatographic separation (Sherman et al., 1973). These measurements of the relative amounts of the iso-l- and iso-2-oytochromes G and the total oytochrome c content permitted the calculation of the absolute amounts of iso-l- and iso-2-oytoohromes G. (d) Preparation

of iso-l-cytochromes

c

The procedure used for large-scale growth of yeast has been described by Prakash et al. (1974). Iso-l-oytoohrome c was prepared by the procedure described by Sherman et al. (1968). Because of the relatively low amounts of iso-1-oytoohrome c in these strains, the following additional purification steps were used. The almost pure samples of iso-l-cytoohrome o from the first ohromatographio gradient were dialyzed and reohromatographed using the same conditions as those of the first ohromatographio purification. The pink eluate was concentrated on a small column of fine Amberlite CG50 cation-exchange resin and the sample was dialyzed in the cold against a O*lO/e (NH&CO3 solution and was applied to a 45 cm x 2.5 cm oolumn of superfine G75 Sephadex with 0.5 drop of mercaptoethanol. The Sephadex had been equilibrated with 0.4 M.-NaCl in a pH 7.5 sodium phosphate buffer, 60 mu, to which 1 ml thioglyoolfl and 10 mg oycloheximide/l had been added. The oolumn was run at 4% with the solution described above, and with a flow rate of 15 ml/h. Two-ml fractions were collected, and the fractions that contained the central portion of the red peak were pooled. The purified oytoohrome c was concentrated with the use of fine resin, dialyzed at 4°C against (NH&CO3 and then freeze-dried. For one strain, L-375, the presence of proteolytio enzymes necessitated the use of an ammonium sulfate precipitation as described in Sherman et al. (1968).

S. W. LIEBMAN,

598

(e) Identification

J. W. STEWART of structural

changes

AND

F. SHERMAN

iv. iso-I-cytochrome

c

Details of the procedures used for total amino acid analysis and for peptide mapping have been published (Stewart et al., 1971; Stewart & Sherman, 1972). Cytochrome c was hydrolyzed with constant-boiling hydrochloric acid, in sealed, evacuated tubes, at lll°C for 20 h. The hydrolysates were dried with a rotary evaporator and analyzed with a Beckman-Spinco amino acid analyzer model 120C. Cytoohrome c was digested in 0.03% ammonium bicarbonate (w/v) with chymotrypsin and with trypsin that was previously treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone. The enzymic digests were lyophilized and were subjected first to electrophoresis on Whatman no. 3MM paper wet with pyridine acetate, pH 6.5, and then to chromatography, in the second dimension, with n-butanol/pyridine/acetic acid/water (15 : 10 : 3 : 12 by vol.). Peptide maps were developed sequentially with a ninhydrin/collidine reagent, with an Ehrlich reagent, and with a Pauly reagent. Details of the preparation and isolation of the principal haem peptide from the peptic digest of iso-l-cytochrome e and from the tryptic digest of the peptic haem peptide, by chromatography on carboxymethyl-cellulose developed with solutions of acetic acid, will be reported elsewhere (Stewart & Sherman, unpublished data). The haem group was cleaved from tryptic haem peptides with mercuric acetate and the haem-free peptides were oxidized with performic acid by procedures to be described elsewhere (Stewart & Sherman, unpublished data). The methods for the partial acetylation of iso-1-cytochrome o with acetic anhydride in sodium acetate, and for the cleavage of partially acetylated cytochrome c with BNPS-skatolet (Fontana, 1972) and of the removal of reagents from the polypeptide products of cleavage will be described elsewhere (Stewart & Sherman, unpublished data). Procedures used for sequential Edman degradation and for thin-layer chromatograpbic identification of NH,-terminal residues are those of Stewart et al. (1971). Dansyl cysteic acid was identified by electrophoresis at pH 12.7 by the method of Gray (1967), using 20 cm x 20 cm thin-layer cellulose plates.

3. Results (a) Altered iso-1-cytochrome

c from cycl ochre mzGtalztssuppressed by SUQ5

The action of XUQ5 was determined from the structures of iso-1-cytochrome c in two different ochre $+ strains, D604-7D (4’ XUQ5 cycl-2) and SL305-7A (zJ+ SUQ5 cycl-72), and in one z/- ochre strain, L-375 (#- iWQ5 ~~~1-72). #+ XUQ5 strains were crossed to each of the cycl-2 and cycl-72 mutants which were previously shown to contain ochre codons correspcmcling, respectively, to amino acid positions 21 and 66 (Stewart

& Sherman,

unpublished

results;

see Sherman & Stewart,

1974) and which

were shown to be suppressible by tyrosine-inserting suppressors (Gilmore et al., 1971; Sherman et al., 1974). As will be described below, although there was variability in the total cytochrome c content of #+ XUQ5 CyGl-8 and #+ XUQ5 cycl-72 meiotic segregants, all of these segregants contained below-normal amounts of cytochrome c but most of them contained a level of cytochrome c that was higher than the amount attributed to only iso-2-cytochrome c. The two segregants D604-7D (#+ XUQS cycl-2) and SL305-7A (I#+ SUQ5 ~~~1-72) that contained typical higher levels of total cytochrome c were chosen for further studies. Chromatographic analysis of cytochrome c from these strains revealed fractions corresponding in position to iso-1-cytochrome c and iso-2qtochrome c. Thus both the increased total amounts of cytochrome c and the presence of cytochrome c at the elution position of iso-l-cytochrome c suggested that #+ SUQ5 was suppressing both cycl-2 and cycl-72. The amino acid composition of protein at the elution position of iso-l-cytochrome c, prepared from the #+ SUQ5 cycl-2 strain (D604-7D) was identical to that of normal 7 Abbreviation

used: BNPS-skatole,

2-(2-nitrophenylsulfonyl)-3-methyl-3-bromoindolenine.

OCHRE

SUPPRESSOR

SERINE TABLE

599

SUBSTITUTIONS

1

Amino acid compositions of iso-1-cytochromes c from normal and suppressed strains

Normal

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half cystine Valine Methionine Isoleuoine Leucine Tyrosine Phenylalanine Tryptophan

16.20 3.78 3.03 11.21 7.74 4.03 9.43 4.27 11.94 7.26 1.97 3.06 1.83 3.83 7.90 4.73 3.91 1

$’ SUQ5 cycl-2 (D6047D) (Residues/molecule) 16.27 3.94 2.79 11.27 7.83 5.00 8.69 4.59 11.49 7.35 2.01 2.85 1.91 3.68 7,74 4.62 3.88 1

a+b’SUQS cycl-7’2 (SL305-7A)

15.86 3.88 3.27 11.33 7.65 4.76 8.39 4.31 11.91 7.21 2.30 3.20 1.87 3.89 7.79 4.64 3.93 1

The normal composition is an average value of 21 normal samples (Stewart et al., 1971). A normal complement of 23 basic residues and 79 acidic and neutral residues excluding tryptophan, half-cystine and methionine was assumed in calculating residues per molecule from mole fractions, in single analyses of 20-h, 6 N-HCl hydrolysatss. Tryptophan was estimated from Ehrlioh-stained peptide maps. Values judged to differ from the normal by 1 or more residues are in bold-face type.

iso-1-cytochrome c except for a single substitution of glutamic acid by serine (see Table 1). Peptide maps of tryptic and chymotryptic digests of the protein (see Fig. 1) were essentially normal for iso-l-cytochrome c. Intragenic revertants of cycl-2 having a substitution of serine for glutamine 21 gave analytical results that were not significantly different from these (Stewart & Sherman, unpublished results). The major haem peptide was isolated from a peptic digest of iso-I-cytochrome c from the z,L+ SUQS cycl-2 strain. This peptide was digested further with trypsin and the resulting haem peptide was isolated, the haem removed with mercuric acetate, oxidized with performic acid and partially sequenced (Table 2). The sequence differs from the normal only by having glutamine 21 replaced by serine. The haemoprotein that eluted from CG50 resin at the position of iso-1-cytochrome ct in the extract of the #+ SUQ5 0~~1-72 strain SL305-7A also was found to have an amino acid composition that differed from that of normal iso-l-cytochrome G by a single substitution of glutamic acid by serine (Table 1). The tryptic peptide map was essentially normal, but the chymotryptic peptide map lacked normal peptides C-8 and C-80x, encompassing residues 62 through 72, and C-8” and C-8”ox, also encompassing residues 65 through 72 (Stewart & Sherman, 1973) (see Fig. 1) and carried two new peptides, which were chromatographically like C-8” and CW’ox but electrophoretically

600

S. W. LIEBMAN,

J. W.

I-

STEWART

AND

F. SHERMAN

Electrophoresis

Origin

Origin

Amino acid

Chymotryptic

4 Origin

digest

Tryptic digest

/Haem Lys-Thr-Arg-Cys-Leu-G/n-Cys-His-Thr-Val-Glu-Lys-Gly-Giy-Pro-His 19 21 I c-3 WT-

,

Asn-Val-Leu-~~p-Asp-G~-Asn-Asn-Met-Ser-Glu-Tyr-Leu-Thr-Asn-Pro-Lys-Lys T-0

C-8’-

--V

I

5’-

C-8’i-

I

C-8”-

FICA 1. Cumulative peptide maps of iso-1-oytocbromes c from normal and suppressed ocbre cycl mutants, and the normal amino acid sequences (Stewart & Sherman, unpublished data) of peptides that are altered in the suppressed strains. Normal, ninbydrin-stained peptide maps (outlined spots) are given by strains D604-7D (I,%+StJQ5 cycl-Z), SL305-7A (4’ SUQ5 ~~~2-72) and L-375 (#- SUQ5 ~~~1-72) with the sole exceptions that strains SL305-7A and L-376 lack no-1 spots C-8, C-8”, C-box, C-VOX (the suffix ox signifies derivatives apparently oxidized at the methionyl residue) and have new spots (filled circles) I and 2. In all peptide maps, spots at normal positions reaoted normally with ninhydrinfcollidine, with the Pauly reagent for tyrosine and hi&dine and with the Ebrlich reagent for tryptophan, except for an unusually intense Ehrlich reaction at C8’ for SL305-7A and L-375. New spots 1 and 2 gave positive reactions for tyrosine but negative reactions for tryptophan. Lower-case letters designate amino acids as follows: a, lysine; b, glutamine; c, glutamic acid and metbionine sulfoxide; d, serine; e, tyrosine and methionine; f, leucine. were

approximately

only

two-thirds as mobile towards the anode similarity of serine and glutamic of the net negative charge of peptide

In view of the chromatographic view

of the

diminution

normal, expected as a consequence by serine, the altered chymotryptic

as C-8” and C-8”ox. acid (Fig. 1)) and in C-8 to two-thirds of:

ofthe replacement of one of its three acidic residues peptide map is consistent with a single replacement

of glutamic acid 66 at the ochre site (Stewart & Sherman, unpublished results) by serine. Partial acetylation of this altered iso-1-cytochrome c to block the NH,terminus, and cleavage with BNPS-skatole, presumably at the single tryptophan

OCBRE

SUPPRESSOR

SERINE

SUBSTITUTIONS

601

TABLET NH,-terminal

sequencesof peptides from iso-l-cytochromes c

Peptide

Sequence

Genotype

Performic acid oxidized, dehaemed, tryptic haem peptide derived from a peptic haem peptide

Total polypeptide products of reaction of BNPS-skatole with partially acetylated cytochrome c

CYCl

Cys-Leu-Gln-Cys19 21

t,5’ SUQS cycl-2

-cys-Leu-ser-cys---19 21

CYGl

Trp-Asp-Glu-Asn-Asn-MetI35

a)+ SUQ5 cycl-72

Trp-Asp&-A&&-Met-r----

$- SUQS cycl-72

Trp-Asp-G-Asn-Asn65-----

Lines below the residues indicate that phenylthiohydantoins were identified, or in the case of cysteic acid, that no phenylthiohydantoins were detected in the organic phase. Lines above the residues indicate that dansyl derivatives were identified. Tryptophanyl residues are deduced from the speci6city of cleavage by BNPS-skatole (Fontana, 1972). The normal CYCl sequences are from Stewart & Sherman (unpublished results). Altered residues are in italic type.

residue 64, yielded a peptide mixture having the partial NH,-terminal sequence shown in Table 2. Serine 66 replaces normal glutamic acid 66 in iso-1-cytochrome c from the I/I+ SUQ5 cycl-72 strain, the same residue that is altered in intragenic revertants of cycl-72 (Stewart & Sherman, unpublished results). A #- I!YYUQ~ cycl-72 strain was obtained as described in Materials and Methods, section (a). Since fllJQ5 is inefficient in $- strains, the cytochrome c content of the $ISUQ5 cycl-72 meiotic segregants could not be distinguished from the mutant level by spectroscopic examinations of whole cells. However, chromatographic analysis of cytochrome c from one of these strains, L-375 (#- SUQ5 cycl-72), which was grown in large quantity, revealed a small fraction corresponding in position to iso-l-cytochrome c while a comparable level of detection did not reveal any iso-1-cytochrome Gfrom a non-suppressed z,G- cycl-72 mutant. The amount of haemoprotein detected at the iso-l-cytochrome c position from L-375, was less than the amount of iso-2-oytochrome c which emerged later from the cation exchange column. The amino acid composition of the putative iso-l-cytochrome c sample is consistent with slightly impure iso-lcytochrome c, but is inconsistent with iso-2-cytochrome G.The protein was subjected to partial sequence analysis in the same way as the protein from the #+ SUQ5 ~~~1-72 strain. The partial NH,-terminal sequence of the acetylated protein treated with BNPS-skatole revealed a serine residue at position 66, and the otherwise normal iso-1-cytochrome c sequence for residues 64 through 68. (b) EJiciencies of suppression, The ef6ciencies with which SUQ5 acts on several cycl alleles in the presence of the non-Mendelian I/ + amplifier of suppression were estimated by coupling the suppressor

602

S. W.

LIEBMAN,

J. W.

STEWART TABLE

AND

F. SHERMAN

3

E$icie?zcies of suppression

Strain nos

Genotypes

4’ SUQ5 cycl-9

16’ SUQ5 cyc_l-2

SLlll-IB

D604-7D

Experiment no.

SL82-1D

Iso-17

Iso-2t

I 2 AV.

20 26 23

7.6

15.5

1.2-1.7

1 2

94 111 102

78.6

23.4

12-18

21 21 21

<0.4$

20.6


Av.

66 57 62

46.3

15.7

7-11

1 2 AV.

22 22 22

2

1 2

17 21 19

< 1.11

1 2

AV. $‘--

SlJQ5 cycl-2 + cycl-2

I&- SUP7-1

cycl-9

$6’ cycl-2

XL-267

1 2

XG773-9A

D604-28

Av.

“Normal” strains

Iso-1-cytoohromec (% of normal)

Total

Av. $+ SUQ5 cycl-179

Cytochrome c concentration (mg cytochrome c/kg dry wt)

§

-

430-650

20

0.3-0.5

17.9

<0.3$

6-30

100

t The iso-l- and iso-2-cytocbromes c were identified by their elution position during chromatographic separation, and their relative amounts were determined spectrophotometricdly by the procedure of Sherman et al. (1973). # The small amount of protein that eluted at the iso-I-cytoohrome c position could result from a low level of revertants or contaminants. f Data from Sherman & Stewart (1971), Gilmore et al. (1971) and Stewart et al. (1971).

with the various cycl alleles and then determining the amounts of suppressed iso-lcytochromes c in the respective strains. An absolute value with which #+ SUQS acts on the various cycl nonsense mutations cannot be given because of the variability of cytochrome c content among different suppressed strains. This variability is probably due to numerous modifier genes that affect the level of suppressed iso-1-oytochrome c. Even in normal strains, the level of iso-1-cytochrome c varies coasiderably (see Table 3). Therefore, the range of the suppression efllciencies was estimated from the spectroscopic examinations of a large number of strains from several pedigrees. Although these estimates are sometimes complicated by the variable background of iso-2qtochrome c (see Table 3), several distinct patterns emerged. As expected, #+ SUQS did not appear to act on the amber mutation cycl-179, nor on the ochre allele cycl-9, but did generally suppress the ochre alleles cycl-2 and cycl-72 at a moderate level. In contrast, z,- SUQ5 didnot appear to act on cycl-72 or cycl-2.

OCHRE

SUPPRESSOR

SERINE

SUBSTITUTIONS

603

C

I i40

560

580

600

'620

Wavelength (nm)

FIG. 2. Low temperature (- 19O’C) spectrophotometric rectordings of intact yeast cells, demonstrating suppression of the ochre alleles cycl-9 and cycl-2 by SUQS. Shown are: #+ GYOl, the normal strain SLlll-1lC; 4’ cycl-2 SUQS, the efficiently suppressed mutant strain D604-7D; $+ oycl-9SUQ5, the inefficiently suppressed mutant strain SLlll-11B; and 4’ cycl-2, the mutant D604-2A, devoid of iso-1-cytochrome c. The absorption maxima of cytochromes a*a3, b, c1 and c are found at 603 mn, 559 nm, 654 nm, and 547 nm, respectively.

Recordings of the low-temperature spectra of representative strains containing #+ SUQ5 and either cycl-9 or cycl-2 are shown in Figure 2. Also shown for comparative purposes are the spectra of a normal # + CYCl strain and a #+ cycl-2 mutant lacking SUQS. Total cytochrome c contents can be estimated from the height of the absorption peaks at 547 nm. It is evident that the #+ SUQ5 cycl-9 strain contains approximately the same amount of cytochrome c as the unsuppressed #+ cycl-2 mutant and that the z)+ SUQS cycl-2 strain contains a level of cytochrome c which is intermediate between that found in the unsuppressed z/’ cycl-2 mutant and the normal #+ CYCl strain. Several representative strains containing #+ SUQ5 and various cycl nonsense mutations were chosen from which the precise amount of suppressed iso-1-cytochrome c was determined by quantitative extraction and chromatographio analysis. The data are in Table 3. In haploid strains, efficiencies of suppression by $+ SUQS were 12 to 18% for cycl-2, l-2 to 1.7% for cycl-9, and less than 0.1% for the amber mutant, cycl-179. In the 4’ cycl-2 strain lacking SUQS, there was less than 0.3% of the normal amount of iso-1-cytochrome c. When the gene dosage of SUQ5 was reduced by one-half in the #’ diploid strain XL-267, cycl-2 was suppressed with a 7 to 11% efllciency. 40

his1 . ma5

. met8

. pet11

. trr1

. km134

-----

f

I

his4

cdc2

horn3 hi81

(serl)

I

ade3

pet3

CUP1

pet1 org.4 thrl

E

:

lysl

cdc29 his6

six2

his5

ma3

ade8

trp4

asp1

lsuppl

pet14

horn2 Isup

trpa met5

ilul

Ep

cdcl2

t

: I

X

urn2

XII

asp5 suplhis2) rad5 ROCl gal2

! turn4

%

arol f CM to r

c

can1 SUP

pFq E

ma2 lys9

pet8

rod52 cdc5

rudl

ISUQSI 1yrl IsLIp1sI

I

XVI

: : : : :

f

i) : : : : : :

t

i

i

+

XVII

ph I,2

llLF12

prt2 fl”12

lysl0

met4

F8

t

F6

thil pdx2

giil

Fm.

3. The genetic map of yeast, emphasizing the location of nonsense suppressors. The se&e-inserting suppressor SU4?5 is on chromosome XVI, while the eight tyrosine-inserting suppressors, SUP& SlJP3, SUPI, SUP5, SUPG, SUP’7, SUP8, and SUPll, are on ohromosomes IV, VI, X, XIII and XV, and on fragment 8 (taken from Mortimer & Hawthorne, 1973; Hartwell et al., 1973; Sherman & Lawrence, 1974).

1

lysll

OCHRE

SUPPRESSOR

SERINE

SUBSTITUTIONS

605

The approximate efficiencies with which the tyrosine-inserting ochre suppressors act on several of these cycl alleles, in $- haploid strains, were estimated by low-temperature spectroscopic examinations of a large number of strains from several pedigrees. Like #+ BUQ5, these suppressors in #- strains do not act on the amber mutation cycl-179, act very poorly on cycl-9 and suppress cycl-2 at a moderate level. Similar observations were made previously by Gilmore et al. (1971) who determined quantitatively that the eight tyrosine-inserting suppressors each act with approximately a 10% efliciency on cycl-2. Several of the suppressors were shown to act on cycl-9 with a low efficiency, but no quantitative measurements were made. As part of the present study, a representative strain bearing the tyrosine-inserting ochre suppressor XUP7-1 and cycl-9 mutations was chosen, from which the amount of suppressed iso-l-cytochrome c was determined by quantitative extraction. The data presented in Table 3 shows that SUP7-1 suppresses cycl-9 with a O-3 to O*5o/oe%ciency. (c) Genetic mapping of SUQ5 A close genetic linkage was observed between SUQ5 and tyr7 in the pedigree of a diploid which was heterozygous for led-12, tyr7-1 and SUQ5, but homozygous for the marker Ks5-2. A clear 2 : 2 segregation was observed for XUQ5, which was scored by the suppression of his5-2. Out of 39 tetrads examined, 34 were parental ditypes, 5 were tetratypes and none was a non-parental ditype for the gene pair XUQ5-tyr7; this frequency of recombinant tetratypes and non-parental ditypes indicates a distance of 6.4 CM (centiMorgans) between the XUQ5 and tyr7 loci. Since the tyr7 locus has been previously mapped (Hawthorne & Mortimer, 1968) and is located on chromosome XVI approximately 25 CM from the centromere (see Pig. 3), the linkage distance of 6.4 CM implies that 8UQ5 and tyr7 are on the same arm of the chromosome. In order to determine the sequence of SUQ5 and tyr7 in relation to the centromere, they were both scored for first and second-division segregation in the five tetratype tetrads using the centromere marker led-12. Three tetrads were 6.rst division for XUQ5, none was first division for tyr7 and two were second division for both markers. These data can be explained by the fewest number of crossovers if fXJQ5 is assumed to lie between tyr7 and the centromere.

4. Discussion The simplest interpretation of the action of XUQ5 on the cycl-2 and ~~~1-72mutants is that this suppressor causes the translation of ochre (UAA) codons as se&e. Introduction of SUQ5 into a #’ cycl-2 ochre strain increased the level of iso-l-oytochrome c from less than 0.3% to approx. 15% of the amount found in normal #- CYGl strains. This suppressed protein contained serine instead of the normal glutamine residue 21, at the position controlled by the ochre triplet. Similarly, introduction of SUQ5 into a z/+ cycl-72 ochre strain yielded a comparable elevation of iso-1-cytochrome c content. This suppressed protein carried a serine replacement for the normal glutamic acid residue 66, at the position coded by the ochre triplet. An identical suppressed protein was isolated from a #- XUQ5 ~~~1-72 strain where it was present in a considerably reduced amount. Since no iso-I-cytochrome c was detected in a #- cycl-Y2 strain lacking known suppressors, it is apparent that SUQ5 causes the translation of UAA as a codon for serine in I$- as well as in #+ strains. Insertion of serine in both #I+ and I/- strains, though more abundant in #+ strains, is consistent with Cox’s (1965,197l) hypothesis that Z/+ acts to increase the e&iency of ii’UQ5.

606

S. W.

LIEBMAN,

J. W.

STEWART

AND

F. SHERMAN

The most probable map position of SUQ5 indicates that it is a new genetic locus, between tyr7 and the centromere of chromosome XVI (see Fig. 3). It is no&w&hy that in the presence of the #’ factor, the action of the SUQ5 suppressor on all tested alleles is identical to that described for the ochre suppressor SUP15 (Hawthorne & Leupold, 1974), which also maps near tyr7. Since the relative positions of the SUP15 (Hawthorne & Mortimer, 1968) and SUQ5 suppressors with regard to the centromere and the tyr7 locus have not been determined definitely, it is possible that SUQ5 is a SUP15 allele. Also, the # factor state of the SUPIS-bearing strains was not reported. A definitive determination of the relation between SUQ5 and SUP15 awaits analyses of crosses between strains bearing the respective suppressors. Although there is still no direct experimental proof, it is generally assumed that many yeast nonsense suppressors are caused by mutated tRNA genes. The most compelling supportive evidence is the strong analogy between the phenomena of nonsense suppression in Escherichia coli and yeast (for reviews, see Mortimer & Gilmore, 1968 ; Hartman & Roth, 1973 ; Hawthorne & Leupold, 1974). The finding that amber and ochre tyrosine-inserting dominant suppressors map at the same genetic loci, strongly suggests that these suppressors arise from tyrosine tRNA altered in two different ways (Sherman et al., 1973; Liebman, 1973). Also, Bruenn & Jacobson (1972) have reported chromatographic differences in the tyrosine tRNA extracted from wild type strains and from strains bearing tyrosine-inserting nonsense suppressors, and they suggested that these differences were directly due to mutated tRNA. Nonetheless, it should be mentioned that Kiger & Brantner (1973) failed to demonstrate the suppressing ability of tyrosine tRNA isolated from strains bearing tyrosineinserting nonsense suppressors in an in vitro E. coli protein synthesizing system. We believe that the most likely origin of the SUQ5 suppressor is a mutation in a tRNA gene. The difficulty of predicting which tRNA species is involved is underscored by the finding in E. coli that a single mutation in a tryptophan tRNA causes the insertion of glutamine at the sites of amber codons (Yaniv et al., 1974; 8011,1974). In this regard it is significant to note (see Fig. 3) that the SUQ5 locus is not linked to any of the loci coding for the tyrosine-inserting amber and ochre suppressors (SUP2, SUP3, SUP4, SUPS, SUPG, SUP7, SUP8, SUPll). Thus the tyrosine-inserting suppressors and the se&e-inserting suppressor undoubtedly arise by mutations of different genes which appear most likely to be, respectively, tyrosine tRNA and serine tRNA. Like the eight tyrosine-inserting ochre suppressors (Gilmore et al., 1971), 1,6+SlJQ5 is specific for ochre (UAA) oodons and does not act on amber (UAG) codons. This specificity is demonstrated by the action of #+ SUQ5 on the bona fide o&e alleles cycl-2 and cycl-72 as well as the presumed ochre alleles h&5-2,lysl-1, ade2-1, arg4-17, and trp5-48 (Cox, 1965; Liebman, 1973) and by the inability of 4’ SUQ5 to act on the bom$de amber allele cycl-179 and the presumed amber allele tyr7-1. The specificity of the ochre suppressors distinguishes them from bacterial oohre suppressors which respond to both UAA and UAG codons. The action of the bacterial ochre suppressor is usually explained by the “wobble” hypothesis (Crick, 1966) since the suppressor anticodon is predicted to pair with both the UAA and UAG codons. The inability to find analogous suppressors in yeast has been discussed previously (Sherman et al., 1973). The biochemical basis for the specificity of the yeast ochre suppressors is unknown; however, several hypotheses have been advanced to explain their action (see Gilmore

OCHRE

SUPPRESSOR

SERINE

SUBSTITUTIONS

607

et at. (1971) for complete discussion). The results in this paper restrict the interpretations presented by Gilmore et al. (1971) and others. Ochre-specific suppressors were predicted by Bock (1967) to occur as a result of the mutation of a tyrosine tRNA anticodon into AUA, followed by deamination to IUA. This suppressor, called “topaz” would, according to the wobble hypothesis (Crick, 1966) recognize both ochre and tyrosine codons. The topaz hypothesis is a consistent explanation for the ochre specificity of the tyrosine-inserting suppressors. As predicted, each of these suppressors causes the insertion of tyrosine at UAA but not UAG codons. The action of XUQ5 can also be explained by the topaz hypothesis, but in this case an additional assumption must be made. If the serine anticodon IGA mutated to the topaz anticodon IUA, the resulting suppressor would be expected to insert serine at both ochre and tyrosine codons. Since SUQ5 does not significantly reduce the cell growth rate, it probably does not act as a general missense suppressor causing the insertion of serine in place of tyrosine. Therefore in order to explain the specificity of XUQ5 by the topaz hypothesis, it must be assumed that the e%ciency of the serine-inserting topaz suppressor on tyrosine codons is extremely low. Ochre-specific suppressors, called sepia, were predicted by Gilmore et al. (1971) to occur by modification of the anticodon of a tRNA, to SUA, when S, at the 5’ position of the anticodon, is a derivative of 2-thiouridine. This hypothesis was based on the conclusion of Yoshida et al. (1971) that S, in the 5’ position of the anticodon of a yeast glutamate tRNA pairs with GAA but not with GAG, and it was inferred that this S can pair with only A in the 3’ position of the anticodon of other tRNAs. In terms of this hypothesis, a GUA or AUA antieodon of tyrosine tRNA and a UGA anticodon of serine tRNA can be mutated to UUA and enzymatically converted to SUA, the sepia anticodon of UAA. The yeast ochre suppressors are therefore compatible with the sepia hypothesis. It is worthy of note that ochre-specific suppressors have not been found in E. coli, although E. coli contains a glutamic acid tRNA specific for GAA (Ohashi et al., 1970) and a glutamine tRNA specific for CAA (Polk & Yaniv, 1972), and in both cases a 2-thiouridiue derivative at the 5’ position of the anticodon has been invoked as the probable cause of specificity for A in the third base of the codon. The e&iency of suppression of UAA codons is affected by the ochre codon’s location in the gene. As Table 4 shows, the tyrosine-inserting suppressor, XUP7-1 and the serine-inserting suppressor XUQ5 each acted on the ochre allele cycl-9 with a much lower eflicienoy than they acted on the ochre allele cycl-2. These observations cannot result from incompatibilities of serine or tyrosine replacements, since studies of intragenic revertant proteins have previously established that the insertion of both TABLE

4

Eflciency of suppression of the cycl-9 and cycl-2 ochre mutants o/o Normal Mutant

cycl-9 cycl-2

Amino rtcid position

(b- SUP’I-1

2 21

o-3-0.5 9-14t

(tyrosine)

t Data, from Gilmore et al. (1971).

iso-l I)+ SUQ5

(serine) l-2-1.7 12-18

S. W.

608

LIEBMAN,

J. W.

STEWART

AND

F. SHERMAN

serine and tyrosine at the sites corresponding to the ochre mutations in cycl-9 and cycl-2 result in normal amounts of iso-I-cytochrome G (Stewart et al., 1972; Stewart & Sherman, unpublished results; Sherman & Stewart, 1974). It is particularly interesting to note that both the serine and tyrosine-inserting suppressors exhibited the same pattern of efficiencies of suppression on the ochre codons located at different positions. Similar effects have been noted in the suppression of nonsense mutants of E. coli (Garen et al., 1965) and T4 phage (Yahata et al., 1970) as well as in the phenotypic reversion of amber and ochre mutants of T4 phage (Salser et al., 1969). Perhaps the location of nonsense codons in the messenger RNA affects their ability to be suppressed. Alternatively, specific sequences surrounding nonsense codons may alter the relative affinities of nonsense codons for suppressor tRNA and for release factors. On the basis of limited data from the lysozyme gene of the bacteriophage T4, Yahata et al. (1970) have proposed that nonsense triplets are suppressed with reduced efllciency if they are followed by sequences resembling nonsense codons. However, this proposal is not supported from the comparison of the suppression efficiencies of various cycl nonsense alleles and their deduced surrounding nucleotide sequences, which is shown in Table 5. Thus the nature of the reading context influence on the suppression of nonsense codons remains to be elucidated. TABLE

5

Nucleotide sequencessurrounding ochre (UAA) acridthe e#ciencies of suppression

Mutant

Amino acid position

Deduoed nuoleotide sequence of mutants

Efficiency of suppression (%I

ACU UAA WC

o-3-1*7t

Ammo acid sequence of normal is0 _1

cycl-9

2

cycl-2

21

-~:~-&-~-

cycl-72

66

-Gp-&-A:-

TikGkP;e-

mutants

U C

U C

9-1st

U TJAA AAC

lo-20$

UN UAA UC

CA;

Nuoleotide sequences were deduced from the known amino acid sequences in the normal and revertant proteins (Stewart & Sherman, 1974; Sherman & Stewart, 1973). The amino acid residues shown in italic type are the corresponding sites of the ochre codon. t For the I$- tyrosine-inserting suppressor and the #* SlJQS suppressor (see Table 4). $ Speotrosoopio estimate for $- tyrosine-inserting suppreacors and the 4’ SUQS suppressor.

The phenotypic similarities of zj+ SUQS and zj- class I, set 1 suppressors in their action on both cycl-2 and the set of five nutritional markers, and the failure to detect eilicient suppression by SUQ5 in #-, and by the class I, set 1 suppressors in 4 haploid cells, directly supports the proposal of Gilmore et al. (1971), that acceptable ranges of efficiency of suppression can restrict the selection of eEcient suppressors of the five nutritional markers. The dissimilarities of tyrosine and serine leave hope that

OCHRE

SUPPRESSOR

SERINE

SUBSTITUTIONS

609

the restrictions in amino acids acceptable to the nutritional markers may not be severe ; there seem few, if any, restrictions on amino acids acceptable at the cycl-2 and cycl-72 sites. Thus an extensive search for efficient suppressors of the five nutritional markers in mutants carrying activity modifiers of nonsense suppressors might uncover still other suppressors. Besides the #+ modifier, which elevates suppressor activity, there are known antisuppressors, which diminish the activity of suppressors. Antisuppressors can reduce the activity of SUQZ, an ef&ient ochre-suppressing allele of SUP11 in #strains, to the point of non-suppression of ade2-1, lysl-.2 and his5-2 in $- strains, and to the point of conversion of SUQ2 from a recessive lethal to an ef&ient suppressor in $+ strains (M&ready $ Cox, 1973). Possibly, ~,6-strains with anti-suppressor may be used to uncover ochre suppressors having inherently greater efficiency of action than the class I, set 1 suppressors.

We greatly

appreciate

the excellent

technical assistance provided by MS M. Jackson, We are especially grateful to Dr Brian S. Cox (Oxford University) for providing strains and for helpful discussions and suggestions. This investigation was supported in part by Public Health Service Research grant GM12702 from the National Institutes of Health, in part by grant no. PF959 from the American Cancer Society, and in part by the United States Atomic Energy Commission at the University of Rochester Atomic Energy Project, Rochester, New York, and has been designated U.S. Atomic Energy Commission Report no. 3490.651. Part of this work has been submitted by one of us (S. W. L.) in partial satisfaction of the requirements for the degree of Doctor of Philosophy.

S. Consaul, E. Risen and N. Broekman.

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