BIOCHIM1E, 1975; 57, 49-59.

tRNAS undermethylation in a met-regulatory mutant of Saccharomyces cerevisiae. Colette FESNEAU~ , Huguette de ROBICHON-SzuLMAJSTER t *, A n n y FnADIN a n d Horst FELDMANN * ". • Laboratoire d ' E n z y m o l o g i e du C.N.R.S., 91190 Gif-sur-Yvette (France). • " Inslitut fiir Physiologische Chemie und P h y s i k a l i s c h e B i o c h e m i e der Universitdt 8000 Miinchen 9, Goetheslrasse 33 (Germany). (17-10-1974).

Summary. - - A study of in vivo and in vitro methylation of tRNAs in regulatory mutants affected in methionine-mediated repression (eth2~ eth3, ethl0) has led to the following results : 1) The eth2-2 carrying strain presents a great undermethylation of its tRNAs, of the same order of magnitude as observed during methionine starvation of methionine auxotrophs. 2) This undermethylation leads to a shift of the tRN Amet peak on a BD cellulose column, ' "'III "

while tRNAI~]t peak is unchanged. 3) The study of a double I~utant strain carrying eth2 and met2 mutations has shown that this undermethy]ation is a consequence of the high internal poo] of methionine. 4) Undermethylation unequally affects the different bases and the different tRNA species.

INTR(~DUC'~JON. W h i l e s t u d y i n g the regulation of the b i o s y n t h e sis of m e t h i o n i n e i n S a c c h a r o m y c e s eerevisiae, recessive m u t a t i o n s l e a d i n g to i m p a i r e d methion i n e - m e d i a t e d r e p r e s s i o n have been o b t a i n e d i n at least three i n d e p e n d e n t loci : eth2, eth3, e t h l 0 [1, 2, 3~. The most typical one isolated so far is the n o n s e n s e m u t a t i o n eth2-2 [2]. I n s t r a i n s carr y i n g this m u t a t e d allele the rate of m e t h i o n i n e b i o s y n t h e s i s is i n c r e a s e d r o u g h l y 20 fold a n d p a r t of the m e t h i o n i n e o v e r p r o d u c e d is excreted [4]. Although repressibility by exogenous S-adenosylm e t h i o n i n e [5] is m a i n t a i n e d , m e t h i o n i n e - m e d i a ted r e p r e s s i o n does not occur i n these strains [3]. An u n e x p e c t e d consequence of m u t a t i o n s such as eth2-2, ethl0-1, ethl0-2 resides i n the f i n d i n g that m e t h i o n i n e e n d o g e n o u s l y o v e r p r o d u c e d or exogenously s u p p l i e d does not lead to the i n c r e a s e i n SAM c o n t e n t ~Thieh n o r m a l l y occurs i n wild type cells or in an other regulatory m u t a n t such as eth3-1 [3]. This p e c u l i a r b e h a v i o u r was s h o w n not to be due to i m p a i r m e n t of m e t h i o n i n e adenosyl transferase itself but more likely to be attributable to the p r e s e n c e of i n t e r n a l i n h i b i t o r s of this enzyme or to i m p a i r e d c o m p a r t m e n t a t i o n . O To whom all correspondence should be addressed. (*) We would llke to dedicate this paper to the memory of Hug,uette de Robichon-Szulmajster prematurely deceased in April 1974 while this manuscript was in preparation.

This alternative p o i n t e d to the possibility that u n complete m e t h y l a t i o n s of m a c r o m o l e c u l e s could occur as an i n d i r e c t consequence of some regulatory mutations. Since other e x p e r i m e n t s have led to implicate methionyl-tRNA as one of the regulatory signals acting in m e t h i o n i n e - m e d i a t e d rep r e s s i o n [6, 71 it was thought of special interest to look first at tRNAs and p a r t i c u l a r l y m e t h i o n i n e tRNAs. Although, as p o i n t e d above, i n d i r e c t effects of r e g u l a t o r y m u t a t i o n s were plausible, it was also possible that the first effect of some of these m u t a t i o n s was c o n c e r n e d w i t h i m p a i r e d tRNA methylases, thus l e a d i n g to tRNAmet molecules u n a b l e to p e r f o r m their regulatory function. A study of in vivo a n d in vitro m e t h y l a t i o n of tRNAs i n different strains was then u n d e r t a k e n a n d extended to the i d e n t i f i c a t i o n of u n d e r m e t h y l ated bases, a n d of tRNA species involved. I n fract i o n a t i o n of methionyl-tRNAs, it a p p e a r e d that u u d e r m e t h y l a t i o n leads to a m o d i f i e d chromatog r a p h i c p a t t e r n of tRNA mmet. MATERIALS AND METHODS. STRAINS : The h a p l o i d strains of S a c c h a r o m g c e s cerevisiae used i n this w o r k are listed i n table I. T h e i r origins have been already described [3, 4, 8]. MEDIA

AND

GROWTH

CONDITIONS'

:

Cells

we, re

g r o w n in m i n i m a l s y n t h e t i c m e d i u m YNB (Difco

5,0

C. F e s n e a u et coll.

Yeast Nitrogen Base w i t h o u t a m i n o acids) supplem e n t e d as p r e v i o u s l y described [4] i n order to satisfy the auxotrophies of the strains used. Cells were harvested d u r i n g e x p o n e n t i a l phase of growth by c e n t r i f u g a t i o n a n d w a s h e d twice. tRNAs were extracted i m m e d i a t e l y (see below). F o r methylases, frozen p a c k e d cells were also used. TABLE I. Strains used. G6notypes

Strains 4094- B El0 CJ2t-11 B MY20 199M1 199M1-102 CM83-9A MM8-17B2 MM53-4C MMI01-2B MM230-2D D6

~, ade2, ural ~, ade2, ural, ethl0-1 ~, ade2, eth3-1 :~, ade2, ural, eth4-1 % trpl, arg4, his5, lysl, ade2, leul :~, trp 1, arg4, his5, lysl, ade2, leul, eth2-2 a, his5, eth2-2 a, nral, ade5-7, leu2, ilel, eth2-7 u, arg4, ural, eth2-2 ~: met2. eth2-2 ~, met6, eth2-2 ~, met2, ura (gene number unknown)

PREPARATION OF tRNAs : tRNAs were extracted as described p r e v i o u s l y [7], b u t all o p e r a t i o n s were c a r r i e d out at 4°C. Lately, a few modifications were used : 1) Macaloid (150 mg per 25 ml of i n i t i a l cell suspension) was added a n d was therefore p r e s e n t d u r i n g p h e n o l extraction ; 2) after the ethanol p r e c i p i t a t i o n , the tRNAs were dissolved in tris-HCl buffer 20 mM pH 7.3 a n d dialyzed overnight against the same buffer. W h e n tRNAs m e t h y l a t e d in vitro by a methylase p r e p a r a t i o n were used ( c h r o m a t o g r a p h i c analysis, see further) tRNAs were separated from the i n c u b a t i o n mixture as follo~vs : the i n c u b a t i o n m i x t u r e is deposited on a DEAE celulose c o l u m n (1.3 cm diameter. X 4 cm) e q u i l i b r a t e d w i t h lris-HC1 buffer pH 7.3 20 mM. P r o t e i n s were elnted by the same buffer c o n t a i n i n g 0.25 M NaC1 (following the 0.D. at 2'8,0 nm) a n d the tRNAs were then eluted b y the same buffer c o n t a i n i n g 0.7 M NaCI. The fractions c o n t a i n i n g the t R N A s (as m e a s u r e d by the O.D. at 260 nm) are pooled a n d the tRNAs are p r e c i p i tated by two volumes of ethanol. They are dissolved i n 1 N HC1 before hydrolysis. tRNA c o n c e n t r a t i o n s were calculated O.D. 250 1 cmnm 22.0 for 1 rag tRNA per ml.

taking

PRglaARATION OF tRNA METHYLASES : Cell free extracts were p r e p a r e d as p r e v i o u s l y described [6]. tRNA methylases have been p u r i f i e d accordBIOCHIMIE, 1975, 57, n ° 1.

ing to Svensson et al. [9] c o n s i s t i n g essentially in a s t r e p t o m y c i n p r e c i p i t a t i o n step followed b y an a m m o n i u m sulfate p r e c i p i t a t i o n (taking the 4080 p. cent fraction). This p u r i f i c a t i o n has been c a r r i e d out ei.ther on freshly p r e p a r e d cells or on frozen cells of the same s t r a i n w i t h no noticeable difference of activity b e t w e e n p r e p a r a t i o n s . ASSAY FOR M E T H Y L A T I O N O F tRNA : Incubation mixtures were essentially according to Philipps and Kjellin-Straby [I0]. They contained in 0.5 m l : 50 ~moles Tris-HCl pH 8.0, 5 l~tmoles MgSO4, 0.05 ~moles EDTA, 1 ~tmo]e glutathion, 50 ~moles ammonium acetate, 1 to 1.5 mg of protein from the 40-80 p. cent enzyme fraction, I0 nanomoles (either 14C or 3H-methy]--S-adenosyl-L-methionine (S.A. 50 mCi/mmole) and varying amounts of tRNAs.

It can be noted here that the more the tRNAs are undermethylated in vivo the more incorporation of methyl groups in the in vitro test is observed. P r e l i m i n a r y e x p e r i m e n t s w e r e c a r r i e d out to d e t e r m i n e for each p r e p a r a t i o n of tRNAs the range of c o n c e n t r a t i o n s w h i c h gave p r o p o r t i o n a l i n c o r p o r a t i o n of CH 3 groups versus tRNA c o n c e n tration. At time intervals (usually 30 m i n ) , 50 ~J samples were taken from i n c u b a t i o n m i x t u r e s a n d d r o p p e d into tubes c o n t a i n i n g 0.5 ml o~ cold 0.1 p. cent TCA ill o r d e r to stop the reaction. Then p r o t e i n s and tRNAs were p r e c i p i t a t e d by f u r t h e r a d d i t i o n of 1 nfl of cold 10 p. cent TCA, left 20 m i n in ice, filtered on W h a t m a n glass fiber circles GF/C a n d w a s h e d first w i t h cold 5 p. cent TCA a n d then by cold eth, anol. After d r y i n g , the r a d i o a c t i v i t y of the filters was c o u n t e d i n a scintillation counter ( I n t e r t e c h n i q u e SL 30). Time curves from 0 to 180 rain were usually followed for each tRNA sample a n d values giveni are calculated from plateau values. A control w i t h o u t tRNAs was deduced for final calculation. PROTEIN DETERMINATION: P r o t e i n estimation was c a r r i e d out by the b i u r e t m e t h o d [11] with bovine s e r u m a l b u m i n as reference. ~ H R O M A T O C,R A P H I C

ANALYSIS

OF

METHYLATED

m e t h y l a t i o n in vitro, the tRNAs were h y d r o l y s e d a n d a n a l y s e d by two different methods : BASES

AND

NUCLEOTIDES"

After

1) Acid hydrolysis o[ the tRNA : D e p e n d e n t u p o n the specific activity of the tRNA sample, various quantitites of labelled tRNAs were used and cold c o m m e r c i a l tRNA was added so that in all cases 1 mg total tRNA was

M e t h i o n i n e b i o s y n t h e s i s in S a c c h a r o m y c e s c e r e v i s i a e . submitted to hydrolysis. Hydrolysis was c a r r i e d out a c c o r d i n g to Hildesheim et al. [12] i n 1 ml of 1 N HC1 for 60 m i n at 100°C in a glass stoppered tube. This p r o c e d u r e leads to complete h y d r o l y s i s into free p u r i n e bases and p y r i m i d i n e nucleotides, w i t h no a p p a r e n t degradation of the methylated RNA constituents [13]. After hydrolysis, samples were dried u n d e r v a c u u m in a r o t a r y evaporator and the residues were solubilized in 200 ~ 0.01 N HCA. T h i n layer c h r o m a t o g r a p h y was c a r r i e d out in two different t w o - d i m e n s i o n a l s y s t e m s : System A (as described by Bjork a n d Svensson [14] a n d m o d i f i e d by Hildesheim et al. [12] consists of solvent 1 ( n - b u t a n o l - w a t e r - c o n centrated a m m o n i a ; 80/9/5) and solvent 2 (isoprop a n o l - c o n c e n t r a t e d h y d r o c h l o r i c a c i d - water ; 1 7 0 / 4 1 / 3 9 ) ; System B (as described by Klagsb r u n [15] consists of solvent 1 ( m e t h a n o l - c o n c e n trated h y d r o c h l o r i c a c i d - w a t e r ; 7/2/1) a n d solvent 2 ( n - b u t a n o l - acetic a c i d - w a t e r ; 4/1,/1). UV a b s o r b i n g spots were localized u n d e r UV light a n d radioactive spots were localized after exposure to Kodirex F i h n s (usually 5-7 days for 5,00,0 c p m deposited at the origin). I d e n t i f i c a t i o n of the spots was o b t a i n e d b y t w o - d i m e n s i o n a l c h r o m a t o g r a p h y of k n o w n m i x t u r e s of various m e t h y l a t e d c o m p o u n d s . Essentially the same dist r i b u t i o n s were observed t h a n those described in [14] a n d [15]. The cellulose b e a r i n g the radioactive spots was s c r a p e d a n d c o u n t e r i n a scintillation counter. 2) T2-ribonuclease digestion : 0.75 A.,60-units of in vitro labelled (3H)-tRNA (2 × 106 cpm) were i n c u b a t e d in 10 ~1 of 0.01 M sodium acetate buffer, pH 4.5, together w i t h 2 u n i t s of T2-ribonuclease for 4 hours at 37°C. The digest was applied to a cellulose t h i n - l a y e r plate (20 X 20 cm) a n d c h r o m a t o g r a p h e d on two dim e n s i o n s as described i n [16], u s i n g the following solvent systems : 1) D i m e n s i o n : i s o p r o p a n o l - 5 p. cent a m m o n i u m acetate buffer (pH 3.5) (60:25, v/v) ; 2) D i m e n s i o n : i s o p r o p a n o l - - 1 M s o d i u m acetate (pH 5.5) - - saturated a m m o n i u m sulfate (1:9:40, v / v / v ) . The four non-labelled nucleotides Ap, Up, Gp and Cp were cochromatog r a p h e d as i n t e r n a l markers. As the label was not sufficient to p r o d u c e visible spots after radioautog r a p h y for two weeks (except m5U), r a d i o a c t i v i t y was d e t e r m i n e d by the c o m b u s t i o n method. F o r this purpose, m a t e r i a l from I cm 2 areas i n a n d a r o u n d the positions of m e t h y l a t e d bases was s c r a p p e d off the plates, b u r n t i n the sample oxidizer (Oxymat, I n t e r t e c h n i q u e ) , and the samples counted for r a d i o a c t i v i t y in oxysolve s c i n t i l l a t i o n BIOCHIMIE,

1 9 7 5 , 57, n ° 1.

51

fluid (Zinsser, F r a n k f u r t ) in a Nuclear Chicago scintillation spectrometer (Isocap 300). E L E C T R O P H R E S I S ON P O L Y A C R Y L A M I D E GELS : Gelelectrophoresis of in vitro methylated tRNA (0.50.7 A260-units) was c a r r i e d out in one or two dimensions. F o r the first d i m e n s i o n 10 p. cent a c r y l a m i d e gels, 3 m m thick, similar to those described in reference [17] were employed, but t a k i n g 30 cm for the r u n n i n g and 8 cm for the spacer gel. F o r a c o n t i n u a t i o n in a second dimension, a gel slice (ca. 1 cm) from the first run, was cut out and p o l y m e r i z e d vertically on top of a 20 p. cent p o l y a c r y l a m i d e gel [18]. The same buffer a n d r u n n i n g c o n d i t i o n s were used as for the first d i m e n s i o n , r u n n i n g time was b e t w e e n 48 a n d 96 hours.

F o r a q u a n t i t a t i v e evaluation, the gel was stained w i t h 1-ethyl-2 [3-(1-ethyl naphtol[1,2d]-thiazolin-2-ylidene)-2-methyl p r o p e n y l ] n a p h t o [1,2d]thiazolium b r o m i d e (Eastman-Kodak) [19]. The single spots were cut out w i t h the help of a small r e c t a n g u l a r p u n c h i n g blade (2 × 3 m m wide) a n d the relative a m o u n t s of tRNA estimated by measur i n g the a b s o r p t i o n i n these gel slices w i t h a modified Zeiss s p e c t r o p h o t o m e t e r PL 4 at 578 nm. The a m o u n t of radioactive label was d e t e r m i n e d subsequer~ily by the c o m b u s t i o n method as described above. CHROMATOGRAPHY OF tRNA ON BENZOYLATEDDEAE CELLULOSE : The s t a n d a r d c o n d i t i o n s desc r i b e d i n ref. 20 were used. COMPOUNDS : The following s t a n d a r d s were used to i d e n t i f y the m e t h y l a t e d c o m p o u n d s : N2m e t h y l g u a n i n e , N2-dimethylguanine a n d 1-methylg u a n i n e were a generous gift from Dr. Hildesheim. 5-methyluracil, 5-methylcytosine, 1 - m e t h y l a d e n i n e a n d 7-methylguanine were from Sigma. S-adenosyl-L-methionine h y d r o c h l o r i d e was p u r c h a s e d from Sigma. The m e t h y l labelled S-adenosyl-Lm e t h i o n i n e was p u r c h a s e d from C.E.A., Saclay (3H, 2.3-3 C i / m m o l e a n d 144, 50 m C i / m m o l e ) a n d front A m e r s h a m (3H, 6.8 Ci/mmole). Yeast comm e r c i a l tRNA was p u r c h a s e d from Schwartz BioResearch. T2-ribonuclease : Sankyo Corp., Tokyo. Cellulose t h i n - l a y e r plates : Merck, Darmstadt. Abbreviations : SAM : S-adenosyl-L-methionine. For nucleotides the n o m e n c l a t u r e following IUPAC-IUB c o n v e n t i o n s were used [Europ. J. Biochem. (1970) 15, 203].

C. F e s n e a u et coll.

52 RESULTS.

I. ¢ In vitro >> METHYLATIONOF tRNAs FROM DIFFERENT STRAINS. It has been a l r e a d y d e s c r i b e d that m e t h i o n i n e a ux o t r o p h s , especially those unable to g r o w on TABI~ II.

In v i t r o methylation of tRNAs extracted

from different strains. Strain

Allele number

+

4094-B CJ21-11 B MY20 El0

eth3-1 eth4-1 ethl0-1

199M1 199MI 102 CM83-9A MM8-17B2

et112-2 eth2-2 eth2-7

+

Millimoles CHJmole tRNA

18 7.2 20 86 2.2 120 45 25

Mutant/ wild type

0.96a 1.1 4.7 54 20 1.1

All cells have been collected in exponential phase of growth and tRNAs purified as described under Materials and Methods. tRNA methylases originate from the wild type strain 4094-B. The incorporations, are calculated from plateau values. (a) The incorporation for 4094-B in the same experiment was 7.5.

h o m o c y s t e i n e , u n d e r m e t h y l a t e t h e i r tRNAs d u r i n g m e t h i o n i n e s t a r v a t i o n [21]. S im i la r results h a v e been r e c e n t l y o b t a i n e d in a s u r v e y of m e t h i o n i n e a u x o l r o p h s ; in t h e in vitro test the highest i n c o r p o r a t i o n of m e t h y l groups, r a n g i n g f r o m 130 to 190 millimoles-CH3 p e r mole of tRNA has been o b s e r v e d for m u t a n t s deficient in the last step of m e t h i o n i n e b i o s y n t h e s i s c o r r e s p o n d i n g to gene MET6 (C. Fesneau, u n p u b l i s h e d results). No ¢ relaxed >> strains of yeast b e in g so far available, this is a p p a r e n t l y the m a x i m a l u n d e r m e t h y l a t i o n of tRNAs w h i c h can be e x p e r i m e n t a l l y attained by the classical use of m e t h i o n i n e starvation. H o w ever, i t should be r e m a r k e d that in E. colt relstrains an i n c o r p o r a t i o n only 4 times h i g h e r is o b s e r v e d [22]. Methionine r e g u l a t o r y mutants have been studied. So far, tRNAs h a v e been e x t r a c t e d only f r o m e x p o n e n t i a l l y g r o w i n g cells and c o m p a r e d to t hei r r e l a t e d p a r e n t a l strain. It can be seen in table II that tRNAs f r o m the t w o w i l d t y p e strains s t u d i ed h e r e are not fully m e t h y l a t e d in vivo and can still accept a f ew m e t h y l groups w h e n i n c u b a t e d in vitro w i t h m e t h y l a s e s e x t r a c t e d f r o m the same strain, thus defining the basal level of i n c o r p o r a -

BIOCHIMIE, 1975, 57, n ° 1.

tion. It is i n c r e a s e d by a factor of 5 in s t r a i n carr y i n g the ethl0-1 mutation. Although this is not a great effect it is v e r y r e p r o d u c i b l e . Other mutations, such as eth3-1 and e.th4-1, do not b r i n g an y change in the in vivo m e t h y l a t i o n of tRNAs. H o w e v e r , the greatest u n d e r m e t h y l a t i o n is obtained w i t h eth2-2 b e a r i n g st r ai n s s h o w i n g ratios of 20 to 55 fold w i t h the c o r r e s p o n d i n g w i l d type. It is r e m a r k a b l e that this u n d e r m e t h y l a t i o n is of the same o r d e r of m a g n i t u d e than the one observ ed d u r i n g m e t h i o n i n e s t a r v a t i o n of m e t h i o n i n e a u x o t r o p h s despite the 20 fold h i g h e r pool of met h i o n i n e f o u n d in these m u t a n t strains [3, 4]. It should be n o t i c e d that a n o t h e r h e t e r o a l l e l i c mutation at the locus ETH2, eth2-7, w h i c h does not lead to s u c h a m a r k e d o v e r p r o d u c t i o n of m et h i o n i n e but a c c u m u l a t e s p a r t of it as SAM [23], does not s h o w u n d e r m e t h y l a t i o n of its tRNAs. Since one of the most s t r i k i n g c h a r a c t e r i s t i c s of the eth2-2 m u t a t i o n is the m a i n t e n a n c e of a ]ow pool of SAM even in the p r e s e n c e of high pool of met h i o n i n e [3], it w o u l d seem, at a first glance, that u n d e r m e t h y l a t i o n m i g h t s o m e w h a t result f r o m this l o w pool of SAM. H o w e v e r , it cannot be in a d i r e c t r e l a t i o n s h i p , since, in eth2-2 b e a r i n g strains, the pool of SAM is still s i m i l a r to the one of the c o r r e s p o n d i n g w i l d type strain. T h e r e f o r e , o t h er (may be i n d i r e c t ) consequence(s) of the dist u r b e d r e g u l a t i o n of m e t h i o n i n e b i o s y n t h e s i s s h o u l d c o o p e r a t e w i t h the l o w pool of SAM to b r i n g stmh a c o n s i d e r a b l e effect. II. UNDERMETHYLATION OF t R N A s

IN THE e t h 2 - 2

BEARING STRAINS IS NOT DUE TO h DEFECT IN tRNA METHYLASF~(S).

Results in th, e first line of table III s h o w that extracts f r o m the m u t a n t strain are able to meTABLE III. In v i t r o methylation of tRNAs from an eth2-2 bearing strain using either methglases [rom

different origins or tRNAs from cells grown in different conditions.

Addition to minimal medium

DL-methionine 2 mM L-SAM o.2 mM

Origin of tRNA methylase wild type

mutant

85 75 5

77.5

tP~NA methylases and tRNAs have been prepared as described under Materials and Method's and legend of table II. Methionine or SAM were present from the beginning of the culture. The results are expressed in millimoles CHJmole tP~NA and calculated from plateau values.

M e t h i o n i n e b i o s y n t h e s i s in S a c c h a r o m y c e s thylate in vitro t h e i r o w n u n d e r m e t h y l a t e d tRNAs. Although s e p a r a t i o n a n d s t u d y of the different m e t h y l a s e s has not been u n d e r t a k e n yet, it seems then u n l i k e l y that any of the m a j o r tRNAs m e t h y lases c o u l d be defective in these extracts. By this e x p e r i m e n t w e c a n n o t e x c l u d e the p r e s e n c e in this s t r a i n of one m o d i f i e d tR~NA methylase. W e have verified that the specific a c t i v i t y of the p r e p a r a tion of m e t h y l a s e s from the m u t a n t eth2-2 is comp a r a b l e to the one f o u n d in the p a r e n t a l strain. However, a slight mo.di.fication of one m e t h y l a s e c a n n o t be e v i d e n c e d b y this assay.

cerevisiae.

53

c a r r y i n g the eth2-2 m u t a t i o n r e s p e c t i v e l y w i t h 3H and 14C-methionine. T h e y a r e then c o c h r o m a t o g r a p h e d on a BD cellulose column a n d the

+

H3 c-, "s9

oa

:', "30~

~.~

,

~

i . ~00

200

III. EXOGENOUS SAM RESTORES A WILD-TYPE MESO.

TnVLATION ~VEL iN eth2-2 tRNAs.

*0o

Since it was a l r e a d y k n o w n that exogenously a d d e d SAM is a c t i v e l y c o n c e n t r a t e d b y eth2-2 s t r a i n s (21 n m o l e s / m g w e i g h t after g r o w t h in the p r e s e n c e of 0.15 irrM exogenous SAM [3]), it was e x p e c t e d that exogenous SAM w o u l d also c o r r e c t the m e t h y l a t i o n defect o b s e r v e d for eth2-2 b e a r i n g s t r a i n s g r o w n in m i n i m a l m e d i u m . Last line of table HI shows that i n d e e d u n d e r m e t h y l a t i o n disa p p e a r s in eth2-2 cells g r o w n in the p r e s e n c e of 2 mM SAM. Also as e x p e c t e d , exogenous methionine w h i c h c a n n o t be t r a n s f o r m e d in vivo into SAM in this s t r a i n [3] does not c o r r e c t tRNAs undermethylation. IV. LUMN

CHROMATOGRAPHY OF

AND T H E

METHIONYL

eth2-2

ON

tRNAs

A

BD

FROM

CELLULOS~ THE

WILD

COTYPE

BEARING STRAIN.

W e have a c y l a t e d in vitro the tRNAs e x t r a c t e d from the w i l d t y p e s t r a i n a n d from the s t r a i n ci

OD~O ~

H3

03

150

~ o o 100

so. so

J ~o ~r~hCTtC~S

2~6

Fro. 1 . BD cellulose column chromatography of m e t h i o n y l tRNAs f r o m the wild type and the eth2-2 bearing strain. The tRNAs were aeytated as described in [6]. Strains used : 4094-B (W. T.) and 199M1-102 (eth2-2--O.D. • - - • eth2-2 O--OW.T.

BIOCHIMIE, 1975, 57, n o 1.

so

IOO FRA3TION$

~se

20o

FIG. 2. - - BD cellulose column chromatography o[ m e l h i o n y l tRNAs from the wild type and the eih2-2 bearing strain grown in the presence of 0.2raM SAM. For aeylation and symbols, see legend of figure 1.

results are s h o w n on figure 1. The first p e a k b e i n g eluted (tRNA~.et) is the f o r m y l a t a b l e species of m e t h i o n i n e specific tRNA a n d the s e c o n d p e a k ( t R N Amet R I ) is the n o n - f o r m y l a t a b l e species. It a p p e a r s that the tRNAI~It f r o m t h e s t r a i n c a r r y i n g the eth2-2 m u t a t i o n s is shifted w i t h r e s p e c t to the same species from w i l d t y p e strain, w h i l e no difference a p p e a r s in the c h r o m a t o g r a p h y of tR,N,~ et of both strains. In o r d e r to see if the o b s e r v e d shift w a s r e a l l y due to the u n d e r - m e t h y l a t i o n of tRNAs e x t r a c t e d f r o m the s t r a i n c a r r y i n g the eth2-2 m u t a t i o n , the f o l l o w i n g e x p e r i m e n t w a s done : the s t r a i n carr y i n g the eth2-2 m u t a t i o n w a s g r o w n in the presence of a SAM c o n c e n t r a t i o n p r e v e n t i n g u n d e r m e t h y l a t i o n (0.2 mM) a n d the tRNAs w e r e then e x t r a c t e d , a c y l a t e d in vitro w i t h aH~L-methionine. The results in figure 2 s h o w that, in these c o n d i tions, w h e r e the tRNAs from the eth2-2 b e a r i n g m u t a n t are not u n d e r m e t h y l a t e d , the tRNA~I~t p e a k is e x a c t l y c o r r e s p o n d i n g to the same s p e c i e s ext r a c t e d from w i l d t y p e , thus i n f e r r i n g that the p r e v i o u s l y o b s e r v e d shift w a s due to u n d e r m e thylation. V. THE

IMPAIRED

METHIONIN]~-MEDIATED

RF~PRE,S -

SION IS NOT A C O N S E Q U E N C E OF T H E U N D E R M E T H Y L A TION.

W e have s h o w n that the m e t h y l a t i o n defect a n d the tRNA~Iet shift on a BD cellulose c o l u m n of the

54

C. Fesneau et coll.

t R N A s e x t r a c t e d f r o m t h e e t h 2 - 2 b e a r i n g s t r a i n is c o r r e c t e d b y e x o g e n o u s SAM w h i c h also l e a d s to r e p r e s s e d l e v e l of m e t h i o n i n e b i o s y n t h e t i c en-

t h e r i g h t o n e i.e. t h e m e l h y l a t i o n d e f e c t is o n l y a c o n s e q u e n c e of t h e d i s t u r b e d m e t h i o n i n e b i o s y n thesis.

TABLE ]IV. and homocysteine

tRNAs methylation s g n t h e t a s e r e p r e s s i o n in v a r i o u s s t r a i n s . Homocysteine synthetaseb~

Strain

MM53-4C MM101-2B D6

.Genetic lesion:

I n vitro methylation ~)

eth2-2 cth2-2, met2 reel2

300 7.2 4.9

MMe)

206 216 48 °~

MM + 2 mM DL-Met

Repression p. cent

0 0 83

269 262 8

(a) Expressed in m i l l i m o l e s CH3 per mole of tRNA. tRNA m e t h y l a s e s f r o m wild type s t r a i n (4094-B) and tRNAs have been p r e p a r e d as described u n d e r m a t e r i a l s a n d methods. In vitro l a b e l i n g was carried w i t h 14C-CHa-SAM for MM63-¢C and MM101-2B a n d w i t h H-CHa-SAM for D6. (b)Units : n a n o m o l e s per m i n u t e per mg (dry weight). Activities were determ i n e d in b e n z e n e - t r e a t e d cells as described in [3]. (c) MM ---- Minimal medi,um. 0.5 mM O-aeetyNDL-homoserine was added to m i n i m a l m e d i u m to ensure g r o w t h of s t r a i n s MM101-2B and D6. (d) It is c o m m o n l y observed t h a t s t r a i n s of different origins show some differences in t h e i r 1,eve~ of enzymes.

zymes. Then, two hypotheses can be made : 1) T h e f i r s t e f f e c t of t h e e t h 2 - 2 m u t a t i o n is t h i s u n d e r m e t h y l a t i o n , t h u s l e a d i n g to tRNAmot m o l e c u l e s u n a b l e to p e r f o r m t h e i r r e g u l a t o r y f u n c t i o n , or, 2) t h i s m e t h y l a t i o n d e f e c t is a c o n s e q u e n c e of t h e d i s t u r b e d r e g u l a t i o n of m e t h i o n i n e b i o s y n t h e sis. I n o r d e r to t e s t t h i s a l t e r n a t i v e , w e h a v e stud i e d t h e t R N A m e t h y l a t i o n of a s t r a i n c a r r y i n g a n eth2-2 m u t a t i o n a n d a m u t a t i o n i n t h e s t r u c t u r a l g e n e of h o m o s e r i n e - O - t r a n s a c e t y l a s e (MET2). T h i s s t r a i n b e i n g u n a b l e to s y n t h e s i z e m e t h i o n i n e d o e s n o t a c c u m u l a t e t h i s a m i n o a c i d ; m o r e o v e r , it c a n b e n o t e d t h a t its e n d o g e n o u s m e t h i o n i n e p o o l is o n l y d u e to t h e t r a n s f o r m a t i o n of t h e O - a c e t y l h o m o s e r i n e a d d e d to t h e m e d i u m f o r g r o w t h , a n d is q u i t e c o m p a r a b l e to t h e e n d o g e n o u s p o o l of a wild type strain (unpublished results from this laboratory). Results in table IV show that the t R N A s e x t r a c t e d f r o m t h e s t r a i n MM101-21B c a r r y i n g a m e t 2 m u t a t i o n i n a d d i t i o n to t h e e t h 2 - 2 m u t a t i o n is n o t u n d e r m e t h y l a t e d in vivo. The s a m e v a l u e is o b t a i n e d i n a s t r a i n b e a r i n g o n l y the met2 mutation. Homocysteine synthetase, t a k e n as a t y p e e n z y m e f o r m e t h i o n i n e b i o s y n t h e sis is still n o t r e p r e s s i b l e b y e x o g e n o u s m e t h i o n i n e i n s t r a i n ~ M 1 0 1 - 2 B , as c a n b e p r e d i c t e d b y t h e p r e s e n c e of t h e e[h2-2 m u t a t i o n . So, it s e e m s t h a l t h e p r e s e n c e of t h e m e t 2 gene, p r e v e n t i n g m e t h i d n i n e a c c u m u l a t i o n , p r e v e n t s also t h e u n d e r m e t h y l a t i o n . It s e e m s t h e n t h a t t h e s e c o n d h y p o t h e s i s is BIOCHIMIE, 1975, 57, n ° 1.

VI. UNDERMETHYLATION OF t R N A s IN e t h 2 - 2 BEARING STRAINS. When tRNAs from the mutan.t strain are met h y l a t e d w i t h 3H-CH3-SAM , d i g e s t e d w i t h T 2 - r i b o -

0® @ o

o

2

@

;¢*t~#

F~6. 3. - - T w o - d i m e n s i o n a l thin-layer chromalogram of nucleotides f r o m tRNA after in v i t r o m e t h y lalion. Designation of the spots is given in table V. The h a t c h e d spots r e p r e s e n t t h e f o u r m a j o r nueleotides t h a t were c o e h r o m a t o g r a p h e d w i t h a T2- digest of labeled tRNA.

w ~

Methionine biosynthesis in Saccharomyces cerevisiae. nuclease and s u b m i t t e d to t w o - d i m e n s i o n a l thinl a y e r c h r o m a t o g r a p h y , at least nine r e d i o a c t i v e spots can be o b s e r v e d (~figure 3 and table V). This TABLE ~.

Distribution of SH-methyl groups in minor nucleotides of tRNA after in v i t r o methylation. Spot {')

Nueleotide

1

A G C U msU (?) mTG m~'C Gm --~ m'G m6A m'2G

2 3 4

5 6

7 8 9

10 11 12 13

cpm

27 1 3 2 1 7

0 0 0 0 000 400 900 300 200 100 650

m~G (~)

5 loo

(?)

2 900

Relative amo~1~t oI total label (p. eentl

52.3 08 37 6.4 4.2 2.0 15.0 9.5 5.6

(*). The spot numbers correspond to those of figure 3.

finding .agrees w i t h the p r e v i o u s c o n c l u s i o n that u n d e r m e t h y l a t i o n in the eth2-2 b e a r i n g strain did not o c c u r as the result of a defect in any partic u l a r tRNA m e t h y l a s e but as one of the n u m e r o u s c o n s e q u e n c e s of d e r e p r e s s e d m e t h i o n i n e biosynthesis. Moreover, the label w a s f o u n d to be distributed rather unequally between several methylated nucleotides. T h e highest i n c o r p o r a t i o n rates w e r e f o u n d for mnU, f o l l o w e d by m2G. A small p e r c e n t a g e (0.8 p. cent) of the label w a s f o u n d in an u n k n o w n n u e l e o t i d e (spot 6 in figure 1), 5.6 p. cent of the label w e r e p r e s e n t in spot 13 of figure 1, w h i c h a c c o r d i n g of its c h r o m a t o g r a p h i c p r o p e r t i e s is t h o u g h t to be a r i b o s e - m e t h y l a t e d dinueleotide, not a~taeked by T2-enzyme. m l A is not stable u n d e r the c o n d i t i o n s of o u r analysis a n d w a s f o u n d as m6A in spot 10 of figure 1. The results o b t a i n e d after a c i d h y d r o l y s i s of tR~CAs m e t h y l a t e d in the p r e s e n c e of I4C-CHa-SAM are s h o w n in table VI. Although t h e r e is some q u a n t i t a t i v e v a r i a b i l i t y d e p e n d i n g u p o n the solvent system used, t w o points can be made. 1) Since the h y d r o l y s i s used yields p u r i n e bases and p y r i m i d i n e n u c l e o t i d e s (see Materials and Methods), it is difficult to assess w h i c h p a r t Of the u n d e r m e t h y l a t i o n is due to base or ribose p o s i t i o n s in spots n a m e d msU a n d maC. H o w e v e r , if one assumes that the level of m e t h y l a t i o n s on the ribose m o i e t y is no m o r e than 4.8 p. cent for

BIOCHIMIE,

I975, 57, n o I.

oo

u r i d i n e , and 18.9 p. cen,t for c y t i d i n e of the total m e t h y l a t i o n of these n u e l e o s i d e s in yeast tRNAs [24] it seems that u r a c i l m e t h y l a t i o n is m o r e i n t e n s e l y affected than cytosine m e t h y l a t i o n (table VII). 2) As far as m e t h y l a t e d p u r i n e s are c o n c e r n e d , the situation is even m o r e s t r i k i n g since 11 p. cent of the m2G but only 1.0 p. cent of the m22G are missing. This huge difference can be c o n s i d e r e d as a c o i n c i d e n t p r o o f that the m e t h y l a t i o n s of g u a n i n e in the N 2 p o s i t i o n l e a d i n g to e i t h e r m o n o or d i m e t h y l d e r i v a t i v e s are e n s u r e d by t w o different methylases. Such a c o n c l u s i o n is by no m e a n s TABLE VI.

Distribution among bases of nndermethylations of tRNAs from wild type and mutant strains. Strain Bases

Mutant/ parental

Parental

Mutant

4.5 0.2 O.5 0.3 0.3

105.0 9.3 15.9 6.6 17.7 4.2

23.9 9.3 79.5 13.2 59.0 1.4

1.6

8.0

5.0

8.2

166.7

20.4

3.6 0.7 O.3 0.2

98.0 9.0 30.8 5.7

27.2 13.0 100.0 28.5

0.7

19.0

27.1

0.4 1.6 0.2

7.1 6.2 2.9

17.7 3.9 14.5

7.3

178.7

24.4

Syshm A mU (') mC (') mO-G m~G m~G mTG m~A X

Syslem B mU(') m C C) m~G m~G

mlG t mTG ,nlA X Y

1.0

Parental s t r a i n : 199M1 ; Mutant strain (eth2-2) : 199M1-102. For tRNAs hydrolysis and solvent systems, see Materials and Method's. 14C-GH3-S,AMwas used. Results have been corrected in order to account for loss of tRNAs during the purification which follows the incubation with methylases. Results are expressed in mmoles/ mole tRNA. X and Y correspond to unidentified compounds. (*) Accounting for mSU and mSC respectively and attached 2' O-me-ribose.

s u r p r i s i n g since it w a s s h o w n p r e v i o u s l y that a genetic defect in the N e - d i m e t h y l g u a n i n e tRNA m e t h y l a s e does not affect the a c t i v i t y of the N:-

56

C. F e s n e a u et coll.

monomethylguanine t R N A m e t h y l a s e [10]. O t h e r m e t h y l a t e d p o s i t i o n s i n g u a n i n e s e e m to b e also a f f e c t e d a n d m ' A is o n l y r e d u c e d b y 1 p. c e n t . TABLE VII. Calculated relative u n d e r m e t h y l a t i o n of d i f f e r e n t bases / o r the w i l d - t y p e a n d eth2-2 t R N A s .

Bases

P. cent o[ each base ("') Total content (') able to accept me-groups of me-bases mmole/mole tRN h )arental straitJ mutant strain

m:'U m"C m~G m.~G m~G mTG m~A

960 --1- 48 (**) 960 ~- 224 (**) 280 56O 800 280 720

0.36 0.06 0.10 0 03 0.04 0.17 0.05

9.75 0.76 11.0 1.02 2.02

4832

0.15

3.7

1.50

0.99

(*) R e s u l t s cal,culated f r o m [24]. (**) 2,-0'-ribose h a s been accounted w i t h p y r i m i d i n e s and in t h e t o t a l since i t is a u t o m a t i c a l l y included (due to the acid h y d r o l y s i s used) in our e s t i m a t i o n s of mSU a n d mSC. (***) N u m b e r s are t a k e n f r o m solvent B system except for m l G a n d mTG w h i c h are b e t t e r d e t e r m i n e d in solvent A (see t a b l e IV). Spots of u n k n o w n n a t u r e have been omitted.

00250m n

I~ t

02

cpm

~

50

5;0

10~

FR ~CTIONS

150

?00

Fie. 4. - - BD cellulose column chromatography of tRNAs extracted f r o m strain 199M1-102 and meth!tlated in vitro. O--O0.D. o--e Radioactivity.

Therefore, alihough roughly only 4 the methyl groups are absent in eth2-2 d i s t r i b u t i o n is v e r y u n e q u a l . O n e h a s a c c o u n t t h e u n e q u a l d i s t r i b u t i o n of BIOCHIMIE, 1975, 57, n ° 1.

p. c e n t of tRNAs, the to t a k e i n these me-

FIG. 5. - - Eleclrophoresis of in vitro methylated tRNA on a 10 p. cent polyacrylamide gel. Des4gnation a n d e v a l u a t i o n of the b a n d s are given in table VIII.

M e t h i o n i n e b i o s y n t h e s i s in S a c c h a r o m y c e s TABLE VIII.

Distribution of .~H-methyl-groups in tRNA bands from 10 p. cenl polyacrylamide gel electrophoresis after in v i t r o methylation of tRNA. Band n • (')

Relative optical density (")

1 (5S RNA) 2 3

1.8 2 5 3.5 2.3 5.3 12 6.3 12.6 5.6 l1.9 9 8.7 7.7 4.8 4.4 5.6 2.5

4

5 6 7 8

9 10 11 12 13 14 15 16 17 18

Relative incorporation rate of 3H-methyl-groups 0.44 4.3 1.8 4.9

4.4 2.5 5.2 5.8 3.4 10.1 3.3 3.9 4.1 3.3 3.2 4.8 12 0.34

4 290 5 660

3 570 I 2 1 2 6

530 570 098 708 861

(**) As calculated from a densitogramm of the stained gel taken at 578 n m with a Zeiss filter photometer PL 4. (*) Evaluation of the bands shown in fignre 5.

~i::i~i~: : i)/ :ii:il!'¸?

':~ fill:) • iJ~¸I~:i! !

cerevisiae.

57

t h y l a t e d bases a m o n g y e a s t t R N A s s p e c i e s . F o r i n s t a n c e [see 24], o n e m22G is f o u n d i n at least six of the y e a s t t R N A s of k n o w n s e q u e n c e , n a m e l y s p e c i f i c t R N A s f o r ~lanine, i s o l e u c i n e , p h e n y l a l a n i n e , s e r i n e I, s e r i n e II a n d t y r o s i n e . A m o n g t h o s e o n l y t w o c o n t a i n , in a d d i t i o n , o n e m2G (tRNA p h e a n d t R N A t y r ) a n d o n e m l G is p r e s e n t o n l y in t R N A p h e a m o n g the six. T h e r e f o r e , a b s e n c e o r s t r o n g r e d u c t i o n of o n e p r e c i s e m e t h y l g r o u p m i g h t be of g r e a t c o n s e q u e n c e f o r a g i v e n "tRNA m o l e c u l e a n d of no c o n s e q u e n c e for a n o t h e r one. One m i g h t t h e n e x p e c t w i d e l y d i f f e r e n t effects of t h e s e m o r e o r less s e l e c t i v e u n d e r m e t h y l a t i o n s o v e r t h e s t r u c t u r e a n d t h e r e f o r e on t h e d i f f e r e n t f u n c t i o n s of a n y g i v e n tRNA. VII.

REPARTITION

OF

THE

UNDERMETHYLATION

AMONG T H E D I F F E R E N T SPECIES OF

tRNAs.

I n o r d e r to s t u d y t h e effect of in vitro m e t h y l a t i o n in i n d i v i d u a l t R N A s p e c i e s f r o m m u t a n t s t r a i n 199M1-102, t h e f o l l o w i n g e x p e r i m e n t w a s c a r r i e d out : t R N A w a s m e t h y l a t e d b y i n c u b a t i o n w i t h a c r u d e e x t r a c t s y s t e m in the p r e s e n c e of 3H-CH3-SAM of h i g h s p e c i f i c a c t i v i t y . T h e n the s e p a r a t i o n of d i f f e r e n t t R N A s p e c i e s w a s p e r f o r med by two methods :

•

Fro. 6. - - Two-dimensional electrophoresis on polyacrylamide gels (see Materials and Methods) Designation and evaluation of the spots are given in table IX. BIOCHIMIE, 1975, 57, n ° 1.

C. F e s n e a u el coll.

58

1) Chromatography on a BD cellulose column. A c o l u m n r u n is s h o w n in figure 4. F r o m the r a d i o a c t i v i t y profile, it can be seen that several species, if not all, w e r e u n d e r m e t h y l a t e d in the m u t a n t strain. 2) Two-dimensional polyacrylamide gel electrophoresis. A r u n on a 10 p. cent gel [17] r e s o l v e d the tRNAs into 16 b a n d s w i t h v a r y i n g i n t e n s i t y (figure 5, table VIII). T h e label w a s n o n - r a n d o m l y d i s t r i b u t e d am o n g these bands, the highest specific activities w e r e f o u n d in bands 4, 7, 8, 10

and 16. T w o - d i m e n s i o n a l e l e c t r o p h o r e s i s on 10 p. cent (1. dimension) and 20 p. cent (2. d i m e n s i o n ) p o l y a e r y l a m i d e gels [18] r e s o l v e d the tRNA population into about 40 spots of d i f f er i n g intensities (figure 6). After staining, optical density and rad i o a c t i v i t y in ev er y spot w e r e d e t e r m i n e d . These data are listed in table IV. As one can see, there is a b e t w e e n 0.4 and 0.6 for most of the co m p o n en t s, but for some of t h e m the specific label is c o n s i d e r a b l y higher. This finding suggests that some specific tR,NAs are s e l e c t i v e l y m e t h y l a t e d in this in vitro system. F o r the moment, it is not possible to m a k e an assessment betw e e n these spots and certain specific tRNAs.

TABLE IX.

Distribation of SH-methyl groups in tRNAs separated by two- dimensional polyacrylamide gel electrophoresis after in v it r o methylalion of tRNA. I

Spot n °

I Optical denstty {*}

cpm

1 2

0.167 0.2 0.11 0.085 0.19 0.2 0.19 0.12 0.195 0.16 0.11 0.16 0.14 0.08 0.15 0.125 0.065 0.129 0.13 0.19 0.O98 0.114 0.07 0.127 0.055 0.068 0.11 0.12 0.085 0.11 0.09 0.1I 0.095 O.O9 0.09 0.09 0.086

134 110 190 34 109 160 116 117 137 211 87 64 52 38 120 238 125 61 172 269 59 73 65 204 76 109 67 135 556 254 31 44 67 25O 96 63 52

3 4

5 6

7 8

9 lO 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3o 31 32 33 34 35 36 37

Relative incorporation rate of 3It-methyl-groups 0.8

0.55 1.7 0.4 0.57 0.8 0.6 0.97 0.70 1.32 079 0.40 0.37 0.47 0.8 1.9 1.92 0.47 1.32 1.42 0.60 0.64 0.93 1.6 1.16 1.6 0.6 1.1 6.5 2.3 0.34 0.4 0.7 2.7 1.06 0.7 0.6

(*) As measured by densitometry at 578 nm in a Zeiss filter photometer PL 4.

BIOCHIMIE, 1975,

57, n ° 1.

DISCUSSION. It has been s h o w n that in the r e g u l a t o r y mutant h i s T of Salmonella typhimurium, the tRNA T M and the tRNA~eu lack a modified base (pseudouridine) in the a n t i c o d o n l~oop and thus are unable to p e r f o r m t h e i r r e g u l a t o r y f u n ct i o n s [25, 26, 27]. As it has been s h o w n in Saccharomyces cereuisiae that the r e p r e s s i o n of m e t h i o n i n e b i o s y n t h e s i s inv o l v e d the f o r m a t i o n of m e t h i o n y l - t R N A met, it was of i n t e r e s t to c l a r i f y the r e l a t i o n s h i p existing betw e e n the tRNAs u n d e r m e t h y l a t i o n and the imp a i r e d methionin.e regulation both p r e s e n t in some of the r e g u l a t o r y mutants st u d i ed in our laboratory. Our first result w as that the m u t at i o n s eth2, eth3 and e t h l 0 do not c o r r e s p o n d to the absence of a tRNA methylase. H o w e v e r , the eth2-2 c a r r y i n g strain presents a h i g h level of u n d e r - m e t h y l a t i o n of its tRNAs. This effect is c o r r e c t e d by exogenous SA,M and not by m e t h i o n i n e . A p a r t i c u l a r study of the chrom a t o g r a p h i c p r o p e r t i e s of m e t h i o n y l - t R N A met has s h o w n that this u n d e r m e t h y l a t i o n leads to a shift of the t R N A ~ t peak on a BD cellulose column. Th e p o ssi b i l i t y that n o n - r e p r e s s i b i l i t y was due to this i m p a i r e d tRNA m e t h y l a t i o n has been excluded by study of the strain M,M101-21B w h e r e rnet h i o n i n e - m e d i a t e d r e p r e s s i o n does not o c c u r despite n o r m a l l y m e t h y l a t e d tRNAs. It is i n t e r e s t i n g to r e m a r k that a strain c a r r y i n g a met6 m u t a t i o n in a d d i t i o n to the eth2-2 m u t a t i o n (MM230-2D) seems to be' m o r e u n d e r m e t h y l a t e d than the eth2-2 met2 m u t a n t (~M101-2B) after g r o w t h in the presence of 0.2 mM m e t h i o n i n e : 42 millilnoles-CrH3 p e r mole of tRNA in MM230-2D an d 7.2 millimolesCH.~ p e r mole of tRNA in MM101-2B. The defect in MM230-2D is in the last step of the m e t h i o n i n e b i o s y n t h e s i s ; this could be m e a n i n g that a product a c c u m u l a t e d in this strain w o u l d be the int e r n a l i n h i b i t o r of methylases. One could p r e d i c t

Methionine

biosynthesis

in Saeeharomyees

that this inhibitor would be either a product of the tetrahydrofolate pathway supplying the met h y l g r o u p of m e t h i o n i n e o r a p r e c u r s o r of m e lhionine before homoeysteine. T h e u n e q u a l d i s t r i b u t i o n of u n d e r m e t h y l a t i o n among the different bases can be explained by d i f f e r e n c e s of a f f i n i t y of t h e t R N A m e t h y l a s e s f o r SAM a n d b y d i f f e r e n c e s i n s e n s i t i v i t y t o w a r d s t h e internal inhibitor(s). A n o t h e r p r o m i s i n g r e s u l t is t h e u n e q u a l u n d e r methylation among the tRNA speeies. The ident i f i c a t i o n of s e v e r a l w i d e l y a f f e c t e d t R N A s p e c i e s w o u l d b e of g r e a t i n t e r e s t f o r t h e s t u d y of t h e c o n s e q u e n c e s of u n d e r m e t h y l a t i o n o n t h e f u n c t i o n s of t h e s e g i v e n t R N A s . Acknowledgements. This investigation was supported b y g r a n t s f r o m the , t h e