Eukaryotic protein synthesis initiation factor 2 (eIF-2) can be phosphorylated on its tl subunit by two wellcharacterised protein kinases, termed the haem-controlled repressor (HCR) and the double-stranded RNAactivated inhibitor (dsI). Phosphorylation of eIF-2 by these kinases is thought to be important in the regulation of peptide-chain initiation. We report the location of the serine residue in the a subunit, which is phosphorylated by both these enzymes. Limited tryptic digestion and subsequent cyanogen bromide treatment of rat liver eIF-2 phosphorylated by HCR yielded one major phosphopeptide. This peptide had the sequence Ile-Leu-Leu-Ser-Glu-Leu-Ser(P)-Arg-Arg .The same major phosphopeptide was obtained from rabbit reticulocyte eIF-2 phosphorylated by HCR or dsl as judged by its behaviour on two-dimensional mapping and reverse-phase chromatography. In all cases the phosphorylated residue was found to be serine-7, and not serine-4, of the above sequence as determined from sequence analysis and by subdigestion of the peptide with Staphylococcus aureus V8 proteinase.Protein synthesis initiation factor-2 (eIF-2) mediates the binding of the initiator tRNA (Met-tRNAi) to the 40s ribosomal subunit during peptide-chain initiation. The activity of eIF-2 is believed to be important in controlling the overall rate of chain initiation and is regulated by phosphorylation of its smallest (a) subunit (reviewed in [l-31). Briefly, phosphorylation of eIF-2a impairs the recycling of eIF-2 which is required to regenerate active eIF-2 from the inactive [eIF-2 . GDP] complexes which are released from the ribosome after each round of chain initiation [4]. This recycling is mediated by an additional initiation factor [5 -91 termed GEF (guanine-nucleotide-exchange factor). This factor promotes the exchange of bound GDP for GTP and yields the active [eIF-2 . GTP] species, which can bind the initiator Met-tRNA, and participate in a further round of chain initiation.The role of the phosphorylation of eIF-2a in the control of translation has been studied extensively in reticulocytes, where haem deprivation rapidly leads to inhibition of chain initiation associated with increased phosphorylation of eIF-2a. This results from the activation of a haem-regulated

The level of the T€F3 mRNA, which encodes the fungal-specific translation elongation factor 3 (EF-3), was measured during the yeast-to-hyphal transition in Candida albicans. In contrast to a previous report, T€F3 mRNA levels were shown to change during dilution into fresh medium, increasing only transiently when dimorphism was induced by either (i) an increase in growth temperature (from 25 "C to 37 "C) combined with the addition of 10% (vh) bovine calf serum to the medium, or (ii) an increase in growth temperature (from 25 "C to 37 "C) combined with an increase in the pH of the medium (from pH 4 5 to 6.5). T€F3 mRNA levels also increased in control cultures under conditions where germ tubes were not formed, but they remained elevated in contrast t o cultures undergoing morphological changes. E F 3 mRNA levels were not significantly affected by heat-shock, but were tightly regulated during batch growth of the yeast form, reaching maximal levels in exponential phase. Therefore, the changes in T€F3 expression that accompany the dimorphic transition in C. albicans appear to reflect the underlying physiological changes that occur during morphogenesis and are not a response to morphogenesis per se. For this reason T€F3 mRNA measurement cannot be used as a loading control in Northern analyses of dimorphic gene regulation. Comparison of TEF3 mRNA levels with the abundance of the EF-3 polypeptide indicated that the synthesis of this essential translation factor might be subject to post-transcriptional regulation.

Clinical isolates of the dimorphic fungus Candida albicans encode a tRNA that, in a cell-free translation system prepared from the yeast Saccharomyces cerevisiae, efficiently translates the amber (UAG) termination codon. Unusually, the efficiency of this UAG read-through in the heterologous cell-free system is not further enhanced by polyamines. The suppressor tRNA is also able to efficiently translate the UAG codon in the rabbit reticulocyte cell-free system and with efficiencies approaching 100% in a homologous (C. albicans) cell-free system. That the suppressor tRNA is nuclear-encoded is demonstrated by the lack of activity in purified C. albicans mitochondrial tRNAs. Finally, UAG suppressor tRNA activity is also demonstrated in three other pathogenic Candida species, C. parapsilosis, C. guillermondii and C. tropicalis. These results suggest that some, but not all, Candida species have evolved an unusual nuclear genetic code in which UAG is used as a sense codon.

As with many other fungi, including the budding yeast Saccharomyces cerevisiae, the dimorphic fungus Candida albicans encodes the novel translation factor, elongation 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 molecular mass of 128 kDa. A polyclonal antibody raised against C. albicans EF-3 also showed cross-reactivity with EF-3 from S. cerevisiae. Similarly, the S. cerevisiae TEF3 gene (encoding EF-3) showed cross-hybridization with genomic DNA from C. albicans in Southern hybridization analysis, 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 polymerase chain reaction-amplified DNA fragment to screen to C. albicans gene library. DNA sequence analysis of 200 bp of the cloned fragment demonstrated an open reading 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. albicans sequence did indeed encode EF-3 was confirmed by demonstrating 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 similarity to EF-3 from S. cerevisiae.

A cell-free poly(U)-dependent translation elongation system from Candida albicans is ATP-dependent due to the presence of an elongation factor 3 (EF3)-like activity. Saccharomyces cerevisiae ribosomes added to a C. albicans postribosomal supernatant (PRS) supported poly(U)-dependent elongation, suggesting that the C. albicans lysate contained a soluble translation factor functionally analogous to the S. cerevisiae translation factor EF-3. The presence of EF-3 in C. albicans was confirmed by Western blotting using an antibody raised against S. cerevisiae EF-3. This antibody was also used to screen a selection of Candida species, all of which possessed EF-3 with molecular mass in the range of 110-130 kDa.