Nucleic Acids Research

Volume 2 number 6 June 1975

Number of genes and base composition of mitochondrial tRNA from Saccharomyces cerevistae J. M. Schneller*, G. Faye**, C. Kujawa**, and A.J. C. Stahl* Laboratoire de Biochimie*, U.E. R. de Sciences pharmaceutiques, Universitd Louis Pasteur, 3, rue de I'Argonne,67083 Strasbourg and Centre de GEn&tique Molculaire du C.N.R.S.** 91190 Gif sur Yvette, France Received 24 March 1975

ABSTRACT

Increasing amounts of mitochondrial [32P] tRNA (4S fraction) were hybridized with mitochondrial DNA of Saccharomyces cerevisiae. At saturation, the calculated number of genes for 4S mitochondrial RNA was 20. Mitochondrial (32P] tRNA eluted from the hydrids obtained either with an excess of tRNA or an excess of DNA showed, after alkaline hydrolysis and chromatography, a G+C content of 28 and 35 p. cent respectively. This last value is similar to that found with the total 4S fraction. The odd nucleotides T (about IT per sequence), TI, hU are present in mitochondrial tRNA. Some sequences may begin with pG. INTRODUCTION

The yeast mit tRNA species chromatograph distinctly from their cytoplasmic counterparts (1, 2, 3). Separated mit valyl-, leucyl-, tyrosyl-tRNA hybridize with mit DNA (4). By now 14 different aminoacyl-tRNA can be hybridized with mit DNA (5). On the other hand, mit leucyl-tRNA synthetase charges more specifically the mit tRNALeu (3). All these results suggest that the primary structure of mit tRNA must be different from that of their cytoplasmic counterpart. As the base composition of nuclear DNA (G + C content = 39 p. cent) and mit DNA (G + C content = 18 p. cent) are very different (6) we studied the overall base composition of mit tRNA. These experiments gave us information about the number of tRNA genes present in mit DNA of Saccharomyces

cerevisiae. MATERIALS AND METHODS

1) The wild type yeast e+ IL8-8C

or the mutant strain

lacking 831

Nucleic Acids Research mit DNA 9. IL8-8C/H71 (7) was grown in a low phosphate galactose medium containing 10 uCi t32P3/l. Purification of the mito-

chondria and extraction of the mit RNA, followed by a sucrose gradient sedimentation, were done according to FAYE et al (8). The high purity of mitochondria was judged from the absence of any contaminating cytoplasmic rRNA. The preparation of mit DNA was as earlier described (9). The 4S fraction from the gradient was then made 0.25 M with NaCl and loaded on a RPC5 column (0.5 x 10 cm) (10), washed with 0.35 M NaCl, 50 mM pH 5.4 NH acetate and eluted in the minimum buffer made 1 M with NaCl. The specific radioactivity was measured in the fractions which had a characteristic RNA spectrum. 2) tRNA-DNA hybridization and elution of the hybridized tRNA: Filters loaded with denatured mit DNA from e+ IL8-8C were incubated at 330C for 23 hours in the presence of mitE32P] 4S RNA in the following mixture: 2 x SSC, NH4 acetate 25 mM pH 5.4, formamide 30 p. cent. The filters were then washed four times in 2 x SSC. One of the washes was in the presence of T1 RNAse (Miles), 2 pg/ml. The dried filters were counted in a thin window SAIP counter. The filters corresponding to the hybridization plateau with RNA excess or those corresponding to the hybridization with DNA excess were boiled three times with 1 ml of distilled water for 5 minutes. 80 to 90 p. cent of the radioactivity bound to the filters were removed. The combined solutions were dried under vacuum, dissolved in 80 p1 water containing 3 to 4 CD260nm of yeast tRNA (Boehringer) and precipitated -with three volumes of ethanol at -200C. The precipitate was finally hydrolysed. RESULTS

1) Purity of the 4S fraction Figure 1 shows that the 4S fraction isolated from sucrose gradients presents only one peak comigrating exactly with a 832

Nucleic Acids Research

cpm

.,~~~~~~~~1 -1000

i

cm

Fig. 1: 7 M

urea, 12 p. cent polyacrylamide gel electrophoresis 10.6 x 1 .0 cm gels (11). : absorbance of added yeast tRNA. ---: mit t32PI 4S RNA. BB: bromophenol blue marker.

on

yeast total tRNA in

a

12

p.

cent polyacrylamide gel.

2) Hybridization of the 4S fraction : The hybridization of E32P) 4S RNA with mit DNA is shown in figure 2. A saturation plateau could be reached. The results are

Fig. 2 : Hybridization of mit 132P3 4S RNA (in pg/ml) with mit D.NA (10 pg per filter). The cpm represent the value of' the hybrid minus blank value. The blank filters gave values lower than 5 p. cent of the DNA filters even with the highest RNA concentrations.

833

Nucleic Acids Research also presented as a SCATCHARD plot (insert) (12). As the specific radioactivity was 12,800 cpm/pg, the assumed MR of tRNA #. 25,000 and the MW of mit DNA 0.5-108 (13), the number of genes for 4S was found to be 20.

3) Base composition : Figure 3 shows an autoradiogram of the chromatography of the hydrolysed L32P] 4S RNA. The hydrolysis was total (no radioactivity at the origin) and the different nucleotides well separated. The spot Y corresponds to the place expected for hU. The spot X, in very low amount, has not been identified but corresponds probably to methylated compounds. The spot pGp represented about 0.5 p. cent in the mitochondrial fractions.

XOrigin

Fig. 3

Autoradiogram on Kodirex films of the mit t32P3 4S RNA hydrolys-ate chromatography according to (14). Hydrolysis was performed for 2 hours in the presence of 15 p. cent piperidine at 1000C croiT1 +T2 RNAse in a 50 mM, pH 4.5, NH4 acetate buffer at 33 C.

Table I reports the base composition of the cytoplasmic tRNA (a), of the total mit t32P) 4S RNA (b), of the 4S RNA eluted from the filters at the hybridization plateau with RNA 834

Nucleic Acids Research excess (c), and from the filters corresponding to the hybridization with mit DNA excess (d). It appears that the G + C content

in mit tRNA is 28 to 35 p. cent, whereas in cytoplasmic tRNA it is 53.5 p. cent. Chemical or enzymatic hydrolysis gave the same results. The odd nucleotides T, 16 and hU are present in the mitochondrial fractions. TABLE I T

V

17.5

1.3

4.1

3.1

26.5

30.5

1.4

3.7

2.4

14

34.5

34.0 (0.7)

2.0

(1.2)

19

30. 5

30. 5 (0. 5)

4. 5

(-

G

C

28.5

25

21.0

17.5 Mit 4S RNA Mit 4S RNA eluted from hybrid with excess RNA (2 to 3 ug RNA hybridized to 10 pg + mit DNA in a 13.8 100 p1 mixture) ) Mit 4S RNA eluted from hybrid with excess DNA (0.1 pg RNA hybridized to 50 pg e+ mit DNA in a 200 pl mixture) 15.0

18

tRNA

Cytoplasmic tRNA

A

U

hU

Comparative base composition of the different tRNA fractions (in mole p. cent). The nucleotide spots were scraped from the plates and radioactivity counted (15). Values in brackets refer to spots which contained less than 30 cpm over blank value. T, V and hU values for cytoplasmic tRNA are from ref. (16). pOp was included in G. DISCUSSION

paic.tNA 1) The comigration of the r32P) 4S PRNA with in 7 M urea, 12 p. cent polyacrylamide gels points out that cytoplasmic and mit tRNA may be of similar sizes. This is in contrast with the report of DUBIN et al. indicating that BHK21 mit tRNA is shorter than the cytoplasmic tRNA (17). This electrophoresis shows also that there is neither lower nor faster migrating material in this 4S RNA. However the problem of a SS RNA comigrating with the 4S remains open. It seems that this hypothesis is unlikely since a 2.4 p. cent polyacryla835

Nucleic Acids Research mide gel electrophoresis of rRNA extracted from mitochondrial

ribosomes

shows no

4-5S peak (8). We also know that

5S RNA is

unmethylated and it is noticable that there is 1.4 p. cent T in the 4S RNA (table I) which corresponds well to one T per tRNA of 70 to 80 nucleotides in length. If such a 4-5S rRNA exists it can

not exceed 3

per cent

of the 4S RNA material,

assuming equi-

molecular amount of the 3 species of rRNA. Such low not influence

based

on

our

amount would

tRNA hybridization results. This estimation is

the molar ratio between 4S RNA and

each

of the rRNAs

calculated from a 2.4 p. cent polyacrylamide gel electrophoresis of total e+ mit RNA shown in ref. (8). 2) The number of 4S genes in the yeast S. cerevisiae mit DNA is similar twenty)to the value earlier found in S. carlsbergensis and with the same methodological uncertainties already discussed (twenty to twenty five) (18). It must be emphasized that we could reach the saturation plateau without adding any cold mit rRNA because of the good homogeneity of our 4S RNA. 3) The G + C content from 28 to 35 p. cent of the mit tRNA is the lowest reported for tRNA and we think that the primary structures of these tRNA may well have some peculiarities. Higher values have been reported for BHK21 mit tRNA : 38 p. cent (19), HeLa cells (20) and Xenopus (21) : 44 p. cent. Our values, very

so

different from that of the cytoplasmic tRNA, are however

notably higher than the G + C content of mit DNA. This shows that tRNA genes like rRNA genes, are regions from the mit DNA in which the G and C are more abundant. This confirms earlier observations of an intramolecular base composition heterogeneity of the mit DNA as stressed by BERNARDI (9) and CARNEVALI (22). Two different hypotheses may account for the difference of the values found with total 4S RNA (35 p. cent) and 4S RNA hybri-

dized with excess RNA (28 p. cent). First the 4S RNA may contain

cytoplasmic tRNA which would increase the G + C content. Secondly the 4S RNA hybridized with excess RNA corresponds to a

some

stoichiometric amount of the different mit tRNA species. We think

836

Nucleic Acids Research that the last hypothesis may be right, for the G + C content of total 4S RNA (35 p. cent) is the same as the one found for mit tRNA hybridized with excess DNA (34 p. cent) - the ratio between the different species of hybridized tRNA draws nearer to that of the total mit 4S RNA -. In order to estimate the contamination

of mit tRNA by cytoplasmic tRNA during mitochondria isolation we prepared [3H] adenine protoplasts of e IL8-8C/H71 and broke them protoplasts were broken in the in a Waring Blendor. Cold 17,000 g supernatant of the e homogenate and the mitochondria

e

were then isolated. The purified mit 4S showed a 4 p. cent contamination by t3H3 cytoplasmic tRNA. The difference between the base composition of total mit 4S RNA and mit 4S RNA eluted

from hybrid with excess RNA suggests that the different mit tRNA species are not in equimolecular amount, as is also suggested by aminoacylation tests. This difference implies also that there is a heterogeneity in G + C content of the individual species.

The identity of the G + C content of the mit tRNA hybridized in excess DNA with that of the total 4S RNA and the presence of about 20 genes in the mit DNA makes in unlikely that cytoplasmic tRNA takes part in the mitochondrial protein synthesis. However the participation of a few cytoplasmic tRNA species cannot

be completely excluded. This is in contrast with the proposals for cytoplasmic tRNA entry into mitochondria of Xenopus and of HeLa where the number of tRNA genes is respectively 15 and 11

(23, 24). 4) Finally we showed the presence of T. Ii, hU and PGD in the mit tRNA which suggests the probable existence of hU and GTTI loops and of sequences beginning with a pG. As already stressed I T may be present per mit tRNA sequence. The presence of T can also be related to our finding of a m5U tRNA methylase in the yeast mitochondria (unpublished results). Acknowledgements : We thank P.P. SLONIMSKI, Centre de G6n4tique Mol4culaire for giving us the yeast strains, H. FUKUHARA for helpful discussion, J. WEISSENBACH for his gift of T1 + T2 RNAse and J. TROWSDALE for his critical reading of the manuscript. This work was supported by the D6partement de Biologie du 837

Nucleic Acids Research Centre d'Etude Atomique de Saclay and the la Recherche Scientifique et Technique.

D6l4gation G6n4rale &

REFERENCES pseudouridylic Abbreviations: T: thymidylic acid ; acid ; hU: dihydrouridylic acid ; 1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate ; mit tRNA, rRNA, RNA or DNA = mito-

chondrial tRNA, rRNA, RNA or DNA.

1)

2)

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