A systematic nomenclature for new translation initiation factor genes from S. pombe and other fungi.

Eukaryotic translation initiation factors and their corresponding genes have been characterized using biochemical and genetic methods from a variety of different organisms. The designations of the factors relate to their apparent roles in the biochemical process. Many gene names indicate genetic interactions with other genes or the functional attributes used to identify them. On the other hand, progress in systematic sequencing of the genomes of organisms like Saccharomyces cerevisiae and Schizosaccharomyces pombe has revealed many genes homologous to known translation initiation factor genes. The genes defined by the systematic sequencing approach are assigned numerical designations completely unrelated to their biological function. So far there have been publications on only three genes encoding translation initiation factors from Schizosaccharomyces pombe. We therefore see this an an ideal opportunity to propose a systematic and logical nomenclature for genes encoding translation initiation factor genes that can be applied to all further genes of this type that are characterized in this fission yeast.

A 110-kilodalton subunit of translation initiation factor eIF3 and an associated 135-kilodalton protein are encoded by the Saccharomyces cerevisiae TIF32 and TIF31 genes.

Translation initiation factor eIF3 is a multisubunit protein complex required for initiation of protein biosynthesis in eukaryotic cells. The complex promotes ribosome dissociation, the binding of the initiator methionyl-tRNA to the 40 S ribosomal subunit, and mRNA recruitment to the ribosome. In the yeast Saccharomyces cerevisiae eIF3 comprises up to 8 subunits. Using partial peptide sequences generated from proteins in purified eIF3, we cloned the TIF31 and TIF32 genes encoding 135- (p135) and 110-kDa (p110) proteins. Deletion/disruption of TIF31 results in no change in growth rate, whereas deletion of TIF32 is lethal. Depletion of p110 causes a severe reduction in cell growth and protein synthesis rates as well as runoff of ribosomes from polysomes, indicative of inhibition of the initiation phase. In addition, p110 depletion leads to p90 co-depletion, whereas other eIF3 subunit levels are not affected. Immunoprecipitation or nickel affinity chromatography from strains expressing (His)6-tagged p110 or p33 results in the co-purification of the well characterized p39 and p90 subunits of eIF3 as well as p110 and p33. This establishes p110 as an authentic subunit of eIF3. In similar experiments, p135 and other eIF3 subunits sometimes, but not always, co-purify, making assignment of p135 as an eIF3 subunit uncertain. Far Western blotting and two-hybrid analyses detect a direct interaction of p110 with p90, p135 with p33, and p33 with eIF4B. Our results, together with those from other laboratories, complete the cloning and characterization of all of the yeast eIF3 subunits.

Characterization of the p33 subunit of eukaryotic translation initiation factor-3 from Saccharomyces cerevisiae.

Eukaryotic translation initiation factor-3 (eIF3) is a large multisubunit complex that binds to the 40 S ribosomal subunit and promotes the binding of methionyl-tRNAi and mRNA. The molecular mechanism by which eIF3 exerts these functions is incompletely understood. We report here the cloning and characterization of TIF35, the Saccharomyces cerevisiae gene encoding the p33 subunit of eIF3. p33 is an essential protein of 30,501 Da that is required in vivo for initiation of protein synthesis. Glucose repression of TIF35 expressed from a GAL1 promoter results in depletion of both the p33 and p39 subunits. Expression of histidine-tagged p33 in yeast in combination with Ni2+ affinity chromatography allows the isolation of a complex containing the p135, p110, p90, p39, and p33 subunits of eIF3. The p33 subunit binds both mRNA and rRNA fragments due to an RNA recognition motif near its C terminus. Deletion of the C-terminal 71 amino acid residues causes loss of RNA binding, but expression of the truncated form as the sole source of p33 nevertheless supports the slow growth of yeast. These results indicate that the p33 subunit of eIF3 plays an important role in the initiation phase of protein synthesis and that its RNA-binding domain is required for optimal activity.

Characterization of cDNAs encoding the p44 and p35 subunits of human translation initiation factor eIF3.

Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit complex that plays a central role in the initiation of translation. It binds to 40 S ribosomal subunits resulting in dissociation of 80 S ribosomes, stabilizes initiator methionyl-tRNA binding to 40 S subunits, and is required for mRNA binding. eIF3 has an aggregate molecular mass of approximately 600 kDa and comprises at least 10 subunits. The cDNAs encoding eight of the subunits have been cloned previously (p170, p116, p110, p66, p48, p47, p40, and p36). Here we report the cloning and characterization of human cDNAs encoding two more subunits of human eIF3, namely eIF3-p44 and eIF3-p35. These proteins are immunoprecipitated by affinity-purified anti-eIF3-p170 antibodies, indicating they are components of the eIF3 complex. Far Western analysis shows that eIF3-p44 interacts strongly and specifically with the eIF3-p170 subunit, and weakly with p116/p110, p66, p40, and itself. eIF3-p44 contains an RNA recognition motif near its C terminus. Northwestern blotting shows that eIF3-p44 binds 18 S rRNA and beta-globin mRNA. Possession of cloned cDNAs encoding all 10 subunits of eIF3 provides the tools necessary to elucidate the functions of the individual subunits and the structure of the eIF3 complex.

The 10Sa RNA gene of Thermus thermophilus.

The 10Sa RNA gene of Thermus thermophilus was isolated and sequenced. The tRNA-like structure at the 5' and 3' ends and other secondary structure features of the T. thermophilus 10Sa RNA are similar to E. coli 10Sa RNA. A variant of the sequence motif coding for the tag peptide is located in the centre of T. thermophilus 10Sa RNA.

Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5.

Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of approximately 600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNAiMet binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.

Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits. Possible roles in RNA binding and macromolecular assembly.

The mammalian translation initiation factor 3 (eIF3), is a multiprotein complex of approximately 600 kDa that binds to the 40 S ribosome and promotes the binding of methionyl-tRNAi and mRNA. cDNAs encoding 5 of the 10 subunits, namely eIF3-p170, -p116, -p110, -p48, and -p36, have been isolated previously. Here we report the cloning and characterization of human cDNAs encoding the major RNA binding subunit, eIF3-p66, and two additional subunits, eIF3-p47 and eIF3-p40. Each of these proteins is present in immunoprecipitates formed with affinity-purified anti-eIF3-p170 antibodies. Human eIF3-p66 shares 64% sequence identity with a hypothetical Caenorhabditis elegans protein, presumably the p66 homolog. Deletion analyses of recombinant derivatives of eIF3-p66 show that the RNA-binding domain lies within an N-terminal 71-amino acid region rich in lysine and arginine. The N-terminal regions of human eIF3-p40 and eIF3-p47 are related to each other and to 17 other eukaryotic proteins, including murine Mov-34, a subunit of the 26 S proteasome. Phylogenetic analyses of the 19 related protein sequences, called the Mov-34 family, distinguish five major subgroups, where eIF3-p40, eIF3-p47, and Mov-34 are each found in a different subgroup. The subunit composition of eIF3 appears to be highly conserved in Drosophila melanogaster, C. elegans, and Arabidopsis thaliana, whereas only 5 homologs of the 10 subunits of mammalian eIF3 are encoded in S. cerevisiae.

Organization of the Thermus thermophilus nusA/infB operon and overexpression of the infB gene in Escherichia coli.

The structural gene for translation initiation factor IF2 from Thermus thermophilus was identified on the basis of the N-terminal amino acid sequence of intact T thermophilus IF2 and an internal 25 kDa IF2 fragment. A total of 5135 bp was cloned and sequenced, comprising the open reading frames for p15A, NusA, p10A, IF2, p10B and SecD, which may form an operon. There are pronounced similarities between the operon arrangement and primary sequence of the T thermophilus genes and proteins, respectively, and their counterparts from other organisms. The T thermophilus infB gene was expressed to a high level in E coli. Four hundred milligrams of homogenous T thermophilus IF2 were prepared from 60 g of overproducing cells.

Identification and purification of translation initiation factor 2 (IF2) from Thermus thermophilus.

Translation initiation factor 2 (IF2) is one of three protein factors required for initiation of protein synthesis in eubacteria. The protein is responsible for binding the initiator RNA to the ribosomal P site. IF2 is a member of the GTP GDP-binding protein superfamily. In the extreme thermophilic bacterium Thermus thermophilus, IF2 was identified as a 66-kDa protein by affinity labeling and immunoblotting. The protein was purified to homogeneity. The specific activity indicates a stoichiometric IF2-mediated binding of formylmethionine-tRNA to 70S ribosomes. The N-terminal amino acid sequences of the intact protein and of two proteolytic fragments of 25 kDa and 40 kDa were determined. Comparison with other bacterial IF2 sequences indicates a similar domain architecture in all bacterial IF2 proteins.

Conservation and diversity in the structure of translation initiation factor EIF3 from humans and yeast.

Initiation factor eIF3 plays a central role in the initiation pathway, influencing ribosome association, ternary complex binding to 40S subunits, and mRNA binding, in part through an interaction with eIF4F. We are attempting to clone and sequence DNAs encoding the subunits of this complex factor. Mammalian eIF3 comprises 10 subunits; full-length human cDNAs have been cloned for eight of these, and partial clones are in hand for the remaining two. Yeast eIF3 comprises at least seven subunits, with six of the seven genes identified and sequenced. Comparison of eIF3 subunit sequences between human and yeast reveals an unexpectedly large diversity of structure. Surprisingly, comparisons with other sequences in the data base suggest that some of the eIF3 subunits may have functions apart from the eIF3 complex. Work is in progress to use the cloned DNAs as tools for elucidating the structure of eIF3 and its interactions with other initiation factors.