Proc. Nati. Acad. Sci. USA Vol. 80, pp. 6192-6196, October 1983 Biochemistry

Molecular cloning and sequence determination of the nuclear gene coding for mitochondrial elongation factor Tu of Saccharomyces cerevisiae (protein synthesis/tuf gene probe/Southern hybridization/sequence conservation) , YASUKO TSUNETSUGU-YOKOTA, AYAKO NAITO, AND YOSHITo KAZIRO Institute of Medical Science, University of Tokyo, Minatoku, Takanawa, Tokyo 108, Japan Communicated by Severo Ochoa, July 8, 1983

ABSTRACT A 3. 1-kilobase Bgl H fragment of Saccharomyces functions in the mitochondrial translational apparatus (16, 17). cerevisiae carrying the nuclear gene encoding the mitochondrial Translational factors as well as ribosomal proteins in the mi- polypeptide chain elongation factor (EF) Tu has been cloned on tochondria are encoded by nuclear genes, synthesized in cy- pBR327 to yield a chimeric plasmid pYYB. The identification of toplasmic fractions, and transported into the mitochondria (18). the gene designated as tufM was based on the cross-hybridization The translational machineries of mitochondria have been thought with the tufB gene, under low stringency con- to be closer to the prokaryotic ones than to the machinery pres- ditions. The complete nucleotide sequence of the yeast tufM gene ent in eukaryotic cytoplasm (17, 19). However, the organization was established together with its 5'- and 3'-flankdng regions. The of the nuclear for the mitochondrial translational sequence contained 1,311 nucleotides coding for a protein of 437 genes appa- amino acids with a calculated Mr of 47,980. The nucleotide se- ratus, their expression, and the incorporation of the products quence and the deduced amino acid sequence of tufM were 60% into the mitochondria are not well understood. and 66% homologous, respectively, to the corresponding se- In this report, we describe the molecular cloning of the yeast quences of E. coli tufA, when aligned to obtain the maximal ho- mitochondrial EF-Tu gene (tufM) utiljzing hybridization with mology. Plasmid YRpYB was then constructed by cloning the 2.5- the E. coli tufB gene under low stringency conditions. The nu- kilobase EcoRI fragment of pYYB carrying tufM into a yeast clon- cleotide sequence as well as the deduced amino acid sequence ing vector YRp-7. A mRNA hybridizable with tufM was isolated of the tufM gene indicate that the structures of E. coli EF-Tu from the total mRNA of S. cerevisiae D13-IA transformed with and yeast mitochondrial EF-Tu are remarkably conserved dur- YRpYB and translated in the reticulocyte lysate. The mRNA could ing evolution. The mRNA for tufM can be translated in the cell- direct the synthesis of a protein with Mr 48,000, which was im- free system from rabbit reticulocytes and the product has been munoprecipitated with an anti-E. coli EF-Tu antibody but not with identified as mEF-Tu by immunoprecipitation with an anti- an antibody against yeast cytoplasmic EF-la. The results indicate body against E. coli EF-Tu. that the tufM gene is a nuclear gene coding for the yeast mito- chondrial EF-Tu. MATERIALS AND METHODS . Endonuclease EcoRI and T4 polynucleotide ligase The polypeptide chain elongation factor Tu (EF-Tu) promotes were gifts from Takashi Yokota. Other restriction enzymes and a GTP-dependent binding of an aminoacyl-tRNA to the A site T4 polynucleotide kinase were purchased from Takara Shuzo of ribosomes (1). EF-Tu from Escherichia coli consists of a sin- (Kyoto, Japan) or Bethesda Research Laboratories and were used gle polypeptide chain with Mr 43,000, and the primary struc- essentially as recommended by the supplier (except for the use ture comprised of 393 amino acid residues has been deter- of 200 ,g of gelatin per ml instead of bovine serum albumin). mined (2). The protein is encoded by two nearly identical genes E. coli DNA polymerase I (large fragment) was purchased from on the E. coli c