Increased Expression of Saccharomyces cerm - due Translation Elongation Factor la Bypasses the Lethality of a TEF 5 N d Allele Encoding Elongation Factor lp

Translation elongation factor 1P (EF-lP) catalyzes the exchange of bound GDP for GTP on EF-la. The lethality of a null allele of the TW5 gene encoding EF-lP in Saccharomyces cerevisiae was suppressed by extra copies of the TW2 gene encoding EF-la. The strains with tefs::TRPl suppressed by extra copies of TEF2 were slow growing, cold sensitive, hypersensitive to inhibitors of translation elongation and showed increased phenotypic suppression of +1 frameshift and UAG nonsense mutations. Nine dominant mutant alleles of TW2 that cause increased suppression of frameshift mutations also suppressed the lethality of teJ5::TRPl. Most of the strains in which tefS::TRPl is suppressed by dominant mutant alleles of TW2 grew more slowly and were more antibiotic sensitive than strains with tefs::TRPl suppressed by wild-type TW2. Two alleles, TW2-4 and TW2-10, interact with tefS::TRPI to produce strains that showed doubling times similar to teJ5::TRPI strains containing extra copies of wild-type TEF2. These strains were less cold sensitive, drug sensitive and correspondingly less efficient suppressors of + 1 frameshift mutations. These phenotypes indicate that translation and cell growth are highly sensitive to changes in EF-la and EF-lP activity. T HE elongation step of protein synthesis involves binding of aminoacyl-tRNA to the ribosomal A site, formation of a peptide bond, and translocation of the newly formed peptidyl-tRNA to the P site (MOLDAVE 1985). The eukaryotic elongation factors EF-1 and EF2 have functions analogous to those of the bacterial elongation factors EF-Tu/Ts and EF-G (MOLDAVE 1985; HINNEBUSCH and LIEBMAN 1991). Yeast require another elongation factor, EF-3, as well (SKOGERSON and WAKATAMA 1976; CHAKRABURTTY and KAMATH 1988). The EF1 complex contains three or four subunits and facilitates binding of aminoacyl-tRNA to the ribosomal A site. The a subunit of EF-1 binds aminoacyl-tRNA in a GTP-dependent manner; this ternary complex then binds to the ribosome ( CARVALHO et al. 1984). The GTP is hydrolyzed to GDP after the cognate aminoacyl-tRNA is bound at the A site. The GDP remains tightly bound to EF-la after this reaction. The p subunit of EF-1 stimulates nucleotide exchange to regenerate EF-la-GTP for the next elongation cycle ( SLOBIN and MOLLER 1978). The function of the y subunit of EF-1 is unknown, although there is evidence that it can stimulate the nucleotide exchange activity of EF-10 (MOLDAVE 1985). Saccharomyces cereuisiae EF-la is an abundant protein, the majority of which is isolated as a monomer (DASMAHAPATRA et al. 1981). In yeast as well as in other eukaryCurresponding author John L. Woolford, Jr., Department of Biological Sciences, 4400 Fifth Ave., Carnegie Mellon University, Pittsburgh, PA 15213. E-mail: [email protected] ’ Present address Department of Molecular Genetics and Microbiology, UMDNJ Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 088545635. Genetics 141: 481-489 (October, 1995) otes, EF-la is also found associated with EF-10 and EF1 y (SAHA and CHAKRAEWRTTY 1986). EF-la in S. cerevisiae is encoded by two unlinked genes, T W l and TW2 (NAGATA et al. 1984). EF-la is essential; a tej1A tePA double null mutation is lethal. Both genes encode identical proteins of 458 amino acids whose sequence is 80% identical to that of EF-la from Artemia salina and humans and 30% identical to Escherichia coli EF-Tu (MERRICK et al. 1990, 1993). The TW5 gene encoding the yeast homologue of EF-lp was isolated by screening a yeast cDNA expression library with antibodies against proteins that associate with calmodulin (HIRAGA et al. 1993). Tm5 is a single copy essential gene. The predicted protein sequence is -50% identical to A. salina and human EF-10. The yeast T W 3 and TW4 genes encode proteins with sequence homology to EF-1 y ( K A ” BOURIS et al. 1993; ~ N Z Y et al. 1994). Disruption of both genes is not lethal and results in no observable defects