Molecular Microbiology (1992) 6(8). 1025-1033 Elongation factor 3 (EF-3) from Candida albicans shows both structural and functional similarity to EF-3 from Saccharomyces cerevisiae D. R. Colthurst,' B. S. Schauder,^ M. V. Hayes^ and M.F. Tuite'* ' BiotogicalLabcratory, University of Kent, Canterbury, Kent CT2 7NJ, UK. ^Microbiology Division, Glaxo Group Research, Greenford Road, Greenford, Middiesex UB6 OHE, UK. Summary As with many other fungi, including the budding yeast Saccharomyces cerevisiae, the dimorphic fungus Candida albicans encodes the novel transia- tion factor, eiongation factor 3 (EF-3). Using a rapid affinity chromatography protocol, EF-3 was purified to homogeneity from C. albicans and shown to have an apparent moiecutar mass of 128 kDa. A poiyclonai antibody raised against C. albicans EF-3 also showed cross-reactivity with EF-3 from S. cerevisiae. Simi- lariy, the S. cerevisiae TEF3 gene (encoding EF-3) showed cross-hybridization with genomic DNA from C. albicans in Southern hybridization anaiysis, demonstrating the existence of a single gene closely related to TEF3 in the C. albicans genome. This gene was cloned by using a 0.7 kb poiymerase chain reaction-amplified DNA fragment to screen a C. albi- cans gene library. DNA sequence analysis of 200 bp of the cloned fragment demonstrated an open read- ing frame showing 51% predicted amino acid identity between the putative C. albicans EF-3 gene and its S. cerevisiae counterpart over the encoded 65-amino- acid stretch. That the cloned C. aibicans sequence did indeed encode EF-3 was confirmed by demon- strating its ability to rescue an otherwise non-viable S. cerevisiae tef3:HIS3 null mutant. Thus EF-3 from C. albicans shows both structural and functional simi- larlity to EF-3 from S. cerevisiae. Introduction The elongation stage of translation in eukaryotes requires two translation elongation factors: EF-1 (a heterotrimer containing the a, p and y subunits), and EF-2 (a single Received 15 November, 1991; revised 6 January, 1992; accepted 8 Jan- uary, 1992. 'For correspondence. Tel. (0227) 764000, exl. 3699; Fax (0227)763912. polypeptide) (Moldave, 1985). The structures of these translation factors are highly conserved across species with, for example, EF-1 from the budding yeast Saccha- romyces cerevisiae showing 81 % amino acid identity with its mammalian counterpart (Brands etai., 1986). There is also conservation of function of elongation factors across these species since S. cerevisiae elongation factors EF-1 and EF-2 are able to support translation elongation on rat- liver ribosomes in v/tro (Skogerson and Engelhard, 1977). However, that the translation elongation mechanism in S. cerevisiae differed from that of rat liver was demonstrated by the failure of EF-1 and EF-2 from rat liver to support translation elongation on S. cerevisiae ribosomes with subsequent fractionation studies implicating the exis- tence of a third elongation factor (termed EF-3) necessary for translation elongation on S. cerevisiae ribosomes (Skogerson and Wakatama, 1976). EF-3 from S. cerevisiae has been purified and charac- terised and shown to be a single polypeptide with an approximate molecular mass of 125 kDa (Dasmahapatra and Chakraburtty, 1981; Uritani and fvliyazaki, 1988a). It exhibits homologous ribosome-dependent ATPase and GTPase activities and stimulates the binding of amino- acyl-tRNA to the ribosome, possibly being involved in the selection of cognate over non-cognate aminoacyl-tRNAs during their transfer from the ribosomal l-site to the ribo- somal A-site (Kamath and Chakraburtty. 1986; 1989; Uri- tani and f\/liyazaki, 1988a,b). That this EF-3-mediated step is essential for the elongation phase of translation has been demonstrated by use of both anti-EF-3 antibod- ies (Dasmahapatra and Chakraburtty, 1981; Hutchison et ai, 1984) and a conditional-lethal (temperature-sensitive) EF-3 mutant (Herrera et ai, 1984; Kamath and Chakraburtty, 1986). The EF-3-encoding gene TEF3 has been isolated and characterized from S. cerevisiae (Qin et ai, 1987; 1990) and shown to encode a 1044-amino-acid polypeptide with the predicted nucleotide-binding sites (Qin et ai, 1990). Intriguingly, the nucleotide-binding sites are duplicated as part of a much larger internally repeated region of about 200 amino acids, and the overall organization of the nucleotide-binding domains places this polypeptide as a member of the ABC (ATP-binding cassette) family of pro- teins (Qin etai, 1990; Colthurst era/., 1991c). Deletion ot the single-copy r£F3 gene leads to inviabitity in haploids.