ORIGINAL PAPER Abstract In an attempt to identify new nuclear genes in- volved in the synthesis and processing of mitochondrial tRNAs, we utilized a multicopy nuclear library to suppress the heat-sensitive phenotype of a Saccharomyces cerevi- siae mitochondrial mutant strain. This strain (Ts 932) is defective in the 3′-end processing of the mitochondrial tRNA Asp transcript. The nuclear genes coding for the mit- ochondrial elongation factor Tuf M and for the mitochon- drial aspartyl-tRNA synthetase have been found to restore the temperature-resistant phenotype and to correct the RNA processing defect. Suppression was effective even when the genes were present on a centromeric plasmid. Key words Mitochondrial tRNA · RNA processing · Tuf M · AspRS M Introduction The mitochondrial genome of yeast carries one copy of each necessary tRNA gene and mitochondrial tRNA bio- genesis requires the presence of several nuclearly encoded factors and of one mitochondrial gene product. These facts open the way to a genetic analysis of tRNAs and tRNA- interacting components. We have taken advantage of this to study a mutation of tRNA Asp which is defective in 3′-end maturation (Zennaro et al. 1989). It is known that mitochondrial tRNAs are released from primary transcripts by processing at the 5′ and 3′ ends. 5′-end processing re- quires mitochondrial RNAse P (Dang and Martin 1993) composed of a nuclear protein and a mitochondrial RNA component, while 3′-end processing is catalyzed by an en- donuclease which has been only partially purified (Chen and Martin 1988). The Saccharomyces cerevisiae Ts 932 mutant exhibits defective growth on glycerol at 36°C which can be ascribed to a C to T transition at position 61 of the tRNA Asp gene, resulting in defective processing of the 3′ end of this tRNA at the non-permissive temperature. In an attempt to better understand the molecular mecha- nism of the deficiency, we searched for multicopy suppres- sors which could (at least partially) restore normal growth. The genes which encode the mitochondrial elongation fac- tor Ef-Tu and the cognate tRNA synthetase have been found to restore the temperature-resistant phenotype and to correct the RNA-processing defect. Materials and methods YPG (1% bactopeptone, 1% yeast extract and 2% glucose), YPGLY (1% bactopeptone, 1% yeast extract and 2% glycerol), YPGAL (1% bactopeptone, 1% yeast extract and 2% galactose), were used as rich culture media for yeast. WO (0.17% yeast nitrogen base, 0.5% am- monium sulphate and 2% glucose) was utilized as a minimal medi- um. All media were supplemented with 2.3% bacto agar (Difco) for solid media, and WO was supplemented with the appropriate nutri- tional requirements according with the phenotype of the yeast strains. As bacterial culture media we used LBA (0.5% yeast extract, 1% bacto-tryptone, 0.5% sodium chloride and 100 μg/ml of ampicillin) or LBAC, which is LBA medium with 10 μg/ml of chlorampheni- col. The yeast strain used to perform the experiments described in this paper is Ts 932/2.12 (Mat a, his3, ade2, leu2, ura3) which pos- sesses a heat-sensitive phenotype at 36°C on glycerol medium. The wild-type nuclear library was constructed in the YEp13 multicopy plasmid. Transformation was obtained by the lithium-chloride pro- cedure of Ito et al. (1983). Mitochondrial RNAs were prepared as described in Baldacci and Zennaro (1982) from wild-type and mu- tant cells grown on YPGAL medium at 36°C. The transformed strain was grown on YPGLY medium at 36°C. Electrophoresis of mito- chondrial RNAs was performed in a 1.5% agarose/6 M urea (par- Curr Genet (1997) 31: 494 – 496 © Springer-Verlag 1997 Received: 23 December 1996 / 17 February 1997 T. Rinaldi · R. Lande · M. Bolotin-Fukuhara L. Frontali Additional copies of the mitochondrial Ef-Tu and aspartyl-tRNA synthetase genes can compensate for a mutation affecting the maturation of the mitochondrial tRNA Asp ORIGINAL PAPER T. Rinaldi () · R. Lande 1 · L. Frontali Pasteur Institute-Cenci Bolognetti Foundation, Department of Cell and Developmental Biology, University of Rome I, I-00185 Rome, Italy M. Bolotin-Fukuhara Laboratoire de Génétique Moléculaire Bat. 400, Université Paris-sud, Orsay, France Present address: 1 Laboratory of Bacteriology and Medical Mycology, Istituto Super- iore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italy Communicated by H. Jacobs