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Biochimica et Biophysica Acta, 520 (1978) 419--427 © Elsevier /North-Hol land Biomedical Press

BBA 99240

N E U R O S P O R A C R A S S A MITOCHONDRIAL TRANSFER RNAs

HANS DE VRIES a, J E N N Y C. DE J O N G E a, J E A N - M A R I E S C H N E L L E R b, R O B E R T P. M A R T I N c, G U Y D I R H E I M E R c and A N D R E J.C. S T A H L b

a Laboratory o f Physiological Chemistry, State University, Bloemsingel 10, Groningen (The Netherlands), b Laboratoire de Biochimie, Facultd de Pharmacie, Universitd Louis Pasteur, 3 rue de l'Argonne, 67083 Strasbourg and c Insfi tut de Biologie Moldculaire et Cellulaire du C.N.R.S., 15 rue Descartes, 67084 Strasbourg (France)

(Received December 28th, 1977)

Summary

Total mitochondrial tRNA from Neurospora crassa was characterized by base composition analysis, one- and two-dimensional gel electrophoreses and reversed-phase chromatography on RPC5. The guanosine + cytidine content was about 43%, as compared to 60% for cytoplasmic tR.NA. The modified nucleoside content was low and about the same as that of total yeast mitochondrial tRNA, though the G + C content is very different. We found ~b, T, hU, t6A, mlG, m:G, m~G. Neither the eukaryotic "Y" base, nor the prokaryotic s4U were present. On two-dimensional polyacrylamide gel electro- pherograms about 25 species were separated. One species for phenylalanine, two for leucine and two for methionine could be located. Neurospora crassa mitochondrial tRNA does not hybridize with yeast mitochondrial DNA.

Introduction

Among the mitochondrial tRNAs, those from Neurospora crassa have been the first to be extensively studied [1--4]. In this report we want to extend the characterization of N. crassa mit tRNAs to the following topics: base composi- tion, an estimation of the number of mit tRNA species by two-dimensional gel- electrophoresis, the presence of iso-accepting tRNAs by RPC5 chromatography of individual tRNA species and the hybridization of mit tRNA with N. crassa and yeast mit DNA.

Abbreviations: PMSF, phenylmethylsul fonyl fluoride; mit: mitochondrial; cyt: cytoplasmic; 1 × SSC, 0.150 M NaC1/0.015 M trisodium citrate.

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Materials and Methods

Materials Diethylpyrocarbonate was from Fluka, DNAase (RNAase-free) from

Worthington, DEAE-cellulose (DE-52) from Whatman, PMSF and cellulose foils from Merck, "stains-all" from Eastman. RPC5 was prepared from Voltalef 300 LD-PL-micro (Ugine-Kuhlmann) and Adogen-464 (Ets A. Arnaud). Radioactive aminoacids were either from Radiochemical Centre, Amersham or from C.E.A., Saclay. 12sI was from N.E.N.

Isolation and purification of N. crassa tRNAs (a) Mitochondrial. Mitochondria were isolated as described earlier [ 5], with

SE1 (0.44 M sucrose, 1 mM EDTA pH 7.5) as the homogenization medium. Further purification was obtained by layering them (suspended in SEs (0.44 M sucrose 5 mM EDTA) on a 10 ml cushion of 1.6 M sucrose, 5 mM EDTA and centrifugation for 10 min at 15 000 rev./min in a Beckman SW27 rotor. The mitochondrial band at the interface was removed, diluted about 5 times with SE~ and pelleted for 20 min at 30 000 × g. The mitochondria were then sus- pended in 10 mM sodium acetate, 10 mM EDTA, 0.1 M sodium thioglycollate, pH 5.0 and, after addition of a drop of diethylpyrocarbonate, lysed with 1% SDS (sodium dodecyl sulfate).

Phenol extraction was then performed twice, followed by ethanol precipita- tion. Ribosomal RNA was removed by precipitation with 2 M NaCl. tRNAs were further purified by t reatment with DNAase (10 pg/ml, 30 min at 20°C), DEAE-cellulose chromatography [6] and Sephadex G100 chromatography [7]. Abou t 750 g of mycelium yielded about 800 pg of mitochondrial tRNAs.

(b) Cytoplasmic. Whole mycelium (about 70 g) was suspended in 300 ml of 0.44 M sucrose, 2 mM EDTA, 10 mM Tris" HC1 pH 7.5, in a Braun kitchen mixer. To the suspension were then added 10 g of acid-washed sand, 5 drops of diethylpyrocarbonate , 10 ml of 20% SDS and 200 ml of a mixture containing phenol/m-cresol/8-hydroxyquinoline (1000 : 140 : 1, v/v). Phenol extraction was then performed at 4°C by mixing for 5 periods of 2 min. Further separa- tion and purification of tRNAs, including DNAase treatment, was as described above.

Isolation and purification of yeast mitochondrial tRNAs was as described before [8].

Base composi t ion analysis. Digestion of tRNAs to nucleosides and 2-dimen- sional chromatography on cellulose foil were performed according to Rogg et al. [91.

Isolation of aminoacyl- tRNA ligases. The yeast mitochondrial enzyme was prepared aceording to Accoceberry et al. [7]. N. crassa mitochondrial enzyme was prepared according to the same method, except that the t reatment with ATP was omitted, that 0.5 mM PMSF (phenylmethylsulfonyl f luo r ide )was present in all steps, and that the DEAE-cellulose purification was omitted. Neurospora "postmitochondria l" enzyme was prepared from a postmicrosomal supernatant by lowering the p H to 5.0 and spinning down the precipitate (15 min at 20 000 ×g). The pellet was dissolved in 20 mM potassium phos- phate, 1 mM MgC12, 20 mM 2-mercaptoethanol, 10% glycerol, pH 7.5. The

421

solution was then passed over a 1 X 5 cm DEAE-cellulose column in the same buffer, and the ligases eluted by raising the phosphate concentration to 250 mM. The protein-containing fractions were pooled and stored at -20°C.

Aminoacylation of tRNAs was performed at pH 8.0 according to Accoceberry et al. [ 71. When N. crassa mitochondrial enzyme was used, 2 mg heparin per ml of incubation mixture was added to prevent degradation by nucleases. For preparative purposes (80 pg of tRNA) we used [3H]leucine (55 Ci/mmol, 4.5 PM final concn.), [3H]phenylalanine (16.6 Ci/mmol, 15 FM final concn.), [ 14C]phenylalanine (522 Ci/mol, 12.5 FM final concn.) or [35S]methionine (69 Ci/mmol, 1 PM final concn.).

RPC5 chromatography was performed as described by Pearson et al. [lo] on a 0.5 X 85 cm column, using linear NaCl gradients of 500 ml.

Polyacrylamide gel electrophoresis. 10% and 20% acrylamide gels in 4 M urea were used. The experimental conditions were as described by Fradin et al.

1111. Radioactive tRNAs. N. crussa mit tRNA was iodinated with Nat2’I as

reported in ref. 12. Isolation of mit DNA and procedure of hybridization are reported in refs. 12

to 14. For the conditions used see legend to Fig. 4.

Results

(1) Base composition of N. crassa tRNAs Total N. crussa mit tRNA has a lower G + C content than cyt tRNA (Table I);

its methylated nucleoside content is two times lower. While m’A, m’G and Gm do not occur in mit tRNAs we find t6A, m’G, m2G, m;G, T, $, hU, I and m5C in trace amount. Neither s4U nor “Y” base can be detected.

10% actylamide, 1M urea

25 20 cm Fig. 1. Electrophoresis of N. crassa mit and cyt tRNAs and yeast mit tRNAs in 10% polyacrylamide, 4 M urea at pH 8.3 [ll]. After electrophoresis for 40 h at 450 V, with Xylene Cyan01 FF (Flub) as tracking dye, the gel was stained with “Stains-all”. Lane A: N. cmsw cyt tRNA: B: N. crassa mit tRNA; C: yeast mit tRNA. The distance from the boundary of the 10% gel with the 6% stacking gel is indicated.

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T A B L E I

N U C L E O S I D E C O M P O S I T I O N O F N. C R A S S A C Y T O P L A S M I C A N D M I T O C H O N D R I A L t R N A s

I n t h e l a s t c o l u m n , t h e e x p e r i m e n t a l v a l u e s h a v e b e e n c o r r e c t e d c o n s i d e r i n g a n d a v e r a g e c o n t a m i n a t i o n o f

1 5 % w i t h c y t t R N A . R e s u l t s axe e x p r e s s e d a s p e r c e n t a g e s o f t h e t o t a l a m o u n t o f n u c l e o s i d e s r e c o v e r e d .

Nucleoside Cyt tRNA Mit tRNA

A 1 6 . 9 0 2 6 . 7 5 U 1 5 . 4 0 2 4 . 2 5

O 3 1 . 4 0 2 1 . 8 5 C 2 4 . 2 0 2 0 . 2 0 m l A 0 . 4 0 - -

t 6 A t r a c e 0 . 2 0

T 1 . 0 0 1 . 2 0

~P 3 . 1 5 1 . 5 5 h U 2 . 7 0 2 . 5 0 m T G 0 . 7 0 - - m l G 1 . 3 5 0 . 5 0

m 2 G 0 . 2 5 0 . 2 5

m 2 G 0 . 5 0 0 . 3 5

G m 0 . 2 0 - -

I 0 . 7 0 0 . 0 4 m S C 1 . 1 5 0 . 0 7 s 4 U * - - , - - , , y , , * * + * * -

G + C 6 0 . 4 5 4 3 . 2 0

m e t h y l a t e d 5 . 5 5 2 . 5 5 n u c l e o s i d e s

* D e t e r m i n e d b y m e a s u r e m e n t o f 3 4 0 r a n a b s o r p t i o n . ** R e f . 4 , a n d o u r o w n r e s u l t s ( n o t s h o w n ) .

F i g . 2 . 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 o f N. crassa m i t o c h o n d r i a l t R N A s . T h e e l e c t r o p h o r e s i s w a s c a r r i e d o u t as d e s c r i b e d b y F r a d i n e t a l . [ 1 1 ] . T h e a r r o w s i n d i c a t e t h e p l a c e o f i s o a c c e p t o r s c a r r y i n g t h e l a b e l l e d a m i n o a c i d s .

423

o

E

c

E

( Jilulda

O

e . e~

~ Z

e~

0 ~

424

(2) Fractionation o f mit tRNAs by polyacrylamide gel electrophoresis (a) One-dimensional electrophoresis. Fig. 1 shows a parallel run of mit and

cyt tRNAs from N. crassa and yeast mit tRNAs. The three patterns obtained differ completely. As noticed in the case of yeast [ 15], the mit tRNAs migrate more slowly than the cyt tRNAs.

(b ) Two-dimensional electrophoresis. Two-dimensional electrophoresis (Fig. 2) fractionates total N. crassa mit tRNA into about 25 major spots, with usually 6 to 10 weaker spots (not visible on the photograph).

(3) Detection o f isoacceptors Among the different spots of the two-dimensional electrophoresis, we identi-

fied the Phe, Met and Leu mit tRNA species by migration of the stabilized [3H]- or [3SS]aminoacyl-tRNA. For charging with leucine and methionine we used a N. crassa mitochondrial ligase preparation, which was completely specific for the mit tRNA. With phenylalanine, the mitochondrial enzymes from either N. crassa or yeast charged mit tRNA and cyt tRNA at the same rate. For this amino acid the yeast enzyme was more active, so this enzyme preparation was used for preparative charging in this case.

Two distinct spots were obtained for each of the three mit aminoacyl-tRNAs (Fig. 2). Distinct peaks were also obtained by RPC5 chromatography (Fig. 3). Two isoacceptors for mit tRNA Met and tRNA Leu and one isoacceptor for mit tRNA Phe are found, which eluted distinctly from the cytoplasmic species. Nevertheless one mit tRNA Phe peak elutes like the cyt tRNA Phe. This is believed to be cyt tRNA contaminating the mit tRNA. On this basis, the amount of con-

o

l o c

o . (3

E (3 . ( J

500(

cpm N.crassa mit tRNA

• ° ~ c r o s . ~ ( o ]

2.5 pg mit DNA of yeast (o) 1000 2pg of Eco/i DNA (x)

_ (~

2 ~s 10 :~ .pg ' [ - t R N A input (per 300 pl medium)

F i g . 4. H y b r i d i z a t i o n o f N . c r a s s a m i t t R N A to N . c r a s s a a n d y e a s t m i t D N A . H y b r i d i z a t i o n w a s p e r - f o r m e d in 3 0 % f o r m a m i d e / 2 × S S C f o r 2 2 - - 2 4 h a t 3 6 ° C . T h e a m o u n t o f m i t D N A p e r f i l t er is 0 . 0 5 p m o l e (2 p g f o r N . c r a s s a m i t D N A , 2 . 5 p g f o r y e a s t m i t D N A ) .

425

tamination should be about 10 to 20%. Note that this contamination is not so well detected for mit tRNA L~u and tRNA Met because of the specificity of the mit enzymes for mit tRNA Le~.

The comparison of the identified spots on two-dimensional electrophoresis in N. crassa and in yeast shows that they are located distinctly (compare Fig. 2 of this report with ref. 15). N. crassa mit tRNA~ he is a minor species. This was concluded from the RPC5 profile and also from the fact that in two-dimen- sional gel electropherograms of cyt tRNA (not shown) the phenylalanine species was located in this area.

(4) Hybr id i za t ion o f N. Crassa m i t t R N A to N. crassa and yeas t m i t D N A A very low level of hybridization is observed in the heterologous hybridiza-

tion when compared to the homologous one {Fig. 4). Using N. crassa mSI-labeled mit tRNA, hybridization with yeast mit DNA is less than 1% of the homologous hybridization after subtraction of the Escherichia coli DNA blanks.

Discussion

Two-dimensional electrophoresis of yeast [15], Locus ta [16] and N. crassa mit tRNA than in cyt tRNA. There might, however, be enough mit tRNA genes the source of tRNAs; several spots may contain more than one tRNAs species [15]. The results indicate the presence of a lower number of isoacceptors in mit tRNA than in cyt tRNA. There might however be enough mit tRNA genes on the yeast mit DNA to allow all codons to be read [15]. In the case of tRNAs Met and tRNAs Leu we could show the presence of two isoacceptors like in yeast [15] and Locus ta [16]. The presence of two N. crassa mit tRNA ~u could not be shown by Epler using RPC2 chromatography [2].

From the data given by Martin et al. [15] it can be concluded that " impor t " of cytoplasmic tRNA species is negligible or even absent. Hence it can be assumed that when cyt tRNA is found in mit tRNA preparations this is caused by contamination. From the RPC5 elution profiles of [3H]Phe-labelled tRNA (Fig. 3), we have estimated the amount of cyt tRNA in our mit tRNA prepara- tion to be about 15%. This amount is low enough to allow a clear interpreta- tion of some features of the whole mit tRNA.

The mit tRNAs investigated so far [14 to 20] have a lower G + C content (30 to 46%) than cyt tRNAs (56 to 62%). There is no clear relationship of mit tRNA G + C content with the position of the corresponding species in evolu- tion: on the one hand yeast [14,21] and Locus ta [16] mit tRNAs have very low [30 to 35%] G + C contents, on the other hand N. crassa, BHK21 cells [17], HeLa cells [18], X e n o p u s [19] or rat-liver [20] mit tRNAs have more than 40% G + C.

Concerning the rare bases however, lower eucaryotes share the presence of T, the absence of mlA, mTG and perhaps also mSC and I since in N. crassa the amount of mSC and I found might be ascribed to 10 to 20% cyt tRNA present in mit tRNA preparations [14,21]. These bases are however present in mit tRNAs from higher eucaryotes, whereas on the contrary the conten t of T is low in this case and perhaps absent [20,22] . A general feature is the absence of the prokaryotic s4U base [8,16] and of the eukaryotic " Y " base [4,8,16]. It

426

should be noticed that the possible absence of I (Table I) should have conse- quences for the decoding capacity of the mit tRNAs, whereas also the number of two leucine isoacceptors is too small to decode all 6 possible leucine codons. Hence the possibility that not all codons occur in mit DNA still cannot be ruled out.

Our results show that N. crassa and yeast [15] mit tRNAs differ in their migration pattern in two-dimensional electrophoresis and in their G + C content . N. crassa mit tRNAs do not hybridize to yeast mit DNA. This indicates the almost complete absence of sequence homology between the tRNAs of these two fungi.

Homologies between mit tRNA from several species have also been checked by hybridization of a single mit tRNA ( tRNA L~u) from the rat with mit DNA from yeast to monkey [ 23]. Good cross-hybridizations only occurred between the rodents systems. The lack of homology between mit tRNAs from species close in the evolution such as N. crassa and yeast or between different vertebrate species contrasts strongly with the situation of cyt tRNAs where it was shown for the initiator cyt tRNA Met that there were no structural differ- ences from fish to man [24] and only very few between N. crassa and yeast [25,26] . These data together suggest that mit tRNA genes have diverged more than those of the procaryotes or those of the eucaryotic nuclei. At present we are investigating the localization of tRNA genes on restriction fragments of the circular N. crassa mit DNA is under investigation and part of the results have been recently presented at an international colloquium [27].

Acknowledgements

The authors wish to thank Professor Dr. A.M. Kroon for encouragement and advice, EMBO for a short-term fellowship to H. de V. to make a 4-week visit to Strasbourg possible, and the Depar tment of Biology of the CEA for aid. This work was supported in part by the Netherlands Foundat ion for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO).

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