Universally conserved translation initiation factors

Bacterial Translation Factor IF-1 and Eukaryotic eIF-1A Are Homologs.

As stated above, despite their general functional similarity—i.e., each facilitates ribosomal subunit dissociation and stabilizes Met-tRNA_i^Met and mRNA binding to the small ribosomal subunit (5, 14, 15)—bacterial IF-1 and eukaryotic eIF-1A have been considered analogs, not homologs (32). However, this is not the case. Homology between the IF-1 and eIF-1A sequences is immediately apparent from profile searches (data not shown) and the alignment of Fig. 1. The archaeal and eukaryotic sequence are on average 38% identical, whereas archaeal and bacterial are 30% and bacterial and eukaryotic are 21%. However, within each of the three major groups, identities are greater than 50%.

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Figure 1

Multiple sequence alignment of bacterial IF-1, eukaryotic eIF-1A, and their archaeal homologs (aIF-1A). Positions in which sequence conservation is >50% identity are highlighted in black. The last line is a consensus of the six S1 motifs found in E. coli ribosomal protein S1: highlighted uppercase type denotes those residues that are also highly conserved in the IF-1/eIF-1A/aIF-1A family, whereas highlighted lowercase type denotes the residues for which a related amino acid occurs in the family. (Dots denote nonconserved positions in the S1 consensus, and dashes denote gaps/insertions of more than one residue.) The horizontal arrows display the positions of the β-strands according to the three-dimensional structure (33). Protein names (and accession number—when different) are as follows: IF1_ECOLI, E. coli IF-1; IF1-SYNEC, Synechocystis sp. IF-1 (EMBL:D90905_47); IF1_BACSU, Bacillus subtilis IF-1; IF1-DEIRA, unpublished ORF from Deinococcus radiodurans; IF1A_METJA, Methanococcus jannaschii aIF-1A; IF1A-METTH, Methanobacterium thermoautotrophicum unpublished ORF; IF1A-ARCFU, Archaeoglobus fulgidus unpublished ORF; YRP2_THEAC, Thermoplasma acidophilum hypothetical protein; IF1A_WHEAT, Triticum aestivum eIF-1A; IF1A_HUMAN, human eIF-1A; IF1A_RABIT, Oryctolagus cuniculus eIF-1A; IF1A_YEAST, Saccharomyces cerevisiae eIF-1A.

Given that the archaeal IF-1A sequences are unique but more similar to their eukaryotic than to their bacterial counterparts, we support the previously proposed renaming of them to archaeal IF-1A (aIF-1A) (32).

A well-known sequence motif, characteristic of bacterial ribosomal protein S1, also has been reported in IF-1 and eIF-2α, as well as in a number of other RNA-binding proteins (34, 35). The (solution) structures for Escherichia coli IF-1 (36) and the S1 domain (33) are built around a five-stranded antiparallel β-barrel, a structure that displays striking resemblance to proteins belonging to the OB (oligonucleotide/oligosaccharide binding) family, many members of which are single-stranded nucleic acid-binding proteins (37). However, the composition of the S1 motif correlates only weakly with the conservation patterns of the IF-1/eIF-1A alignment (see Fig. 1), not only among Bacteria, Archaea, and Eukarya but even within the Bacteria themselves. These conservation patterns would seem, then, to suggest functionality over and above simple nucleic acid binding.

Eukaryotic Initiation Factor eIF-1/SUI1 Occurs in Archaea and Some Bacteria.

Although mammalian eIF-1 is a single subunit factor (38) and yeast SUI1 is one of the eight eIF-3 subunits (17), the two are very similar at the sequence level (59% identity), and both are reported to have similar functions, i.e., stabilizing mRNA and initiator tRNA binding to the 40S ribosomal subunit (16).

As the alignment of Fig. 2 shows, all known archaeal genomes contain homologs of eIF-1/SUI1 (11). A few bacterial examples are known as well [i.e., enteric bacteria (39, 40) and cyanobacteria (41)], although the majority of the known bacterial genomes [i.e., Helicobacter pylori (42), Mycoplasma genitalium (43), M. pneumoniae (44), Treponema pallidum, Borrelia burgdorferi, and Deinococcus radiodurans] contain none.

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Figure 2

Multiple sequence alignment of the eukaryotic SUI1/eIF-1 protein family with their archaeal and bacterial homologs. Protein names (and accession number—when different) are as follows: YCIH_ECOLI, E. coli hypothetical protein; YCIH_SALTY, Salmonella typhimurium hypothetical protein; YCIH_HAEIN, Haemophilus influenzae hypothetical protein; YCIH-SYNEC, Synechocystis sp. hypothetical protein (European Molecular Biology Laboratory accession no. D64003_48); SUI1-METJA, M. jannaschii hypothetical ORF MJ0463 (Protein Information Resource accession no. G64357); YRP1_METVA, Methanococcus vannielii hypothetical protein; SUI1-METTH, M. thermoautotrophicum unpublished ORF; SUI1-ARCFU, A. fulgidus unpublished ORF; SUI1_HUMAN, human SUI1; SUI1_ANOGA, Anopheles gambiae SUI1; SUI1_YEAST, Saccharomyces cerevisiae SUI1; SUI1_ARATH, Arabidopsis thaliana SUI1.

According to Table 1, percent identities between domains are more or less the same, in the 25–30% identity range, a break with the now-familiar pattern of the highest similarity being between archaeal and eukaryotic components of the translation components. Within each major group, the percent identities are greater than 55%, except for the bacterial examples (see Table 1). The 38% identity between the enteric Bacteria and the cyanobacterium questions their orthology. Although it is reasonable to assume that Archaea possess a eIF-1/SUI1 type of function (based on the overall similarity of archaeal and eukaryotic translation initiation), one wonders about the functional significance of the few phylogenetically scattered bacterial examples. Given that the bacterial versions of eIF-1/SUI1 are no more diverged from the archaeal and eukaryotic versions than these are from one another, the idea that this molecule is involved in translation initiation in some Bacteria (despite its being paralogous therein) must be seriously considered and subject to experimental test.

It has been suggested that SUI1 functions in concert with eIF-2 to confine Met-tRNA_i^Met recognition to the AUG initiator codon, because mutants of SUI1 allow tRNA_i^Met to initiate protein synthesis at UUG codons as well (45). Because it is believed that there may be a functional relationship between the eukaryotic and the bacterial start-site selection processes (46), the presence of SUI1 homologs in some bacterial genomes supports a similar start-site recognition mechanisms among Archaea, Eukarya, and some Bacteria.

Archaeal/Eukaryotic Initiation Factor eIF-5A Is Related to Bacterial Elongation Factor EF-P.

Eukaryotic translation initiation factor eIF-5A appears to promote the synthesis of the initial peptide bond in mRNA translation (47) and is unique in being the only known cellular protein that contains the unusual amino acid hypusine, formed by a posttranslational modification of a specific lysine residue (48). This (modified) protein has been identified in all Eukarya and Archaea so far analyzed, but it appears to be absent from Bacteria (49). However, a similar facilitating function may be performed in Bacteria by translation factor EF-P (50).

Fig. 3 shows that, despite the lack of hypusine in the bacterial case, bacterial EF-P and eukaryotic/archaeal eIF-5A are homologs. Although sequence similarity between the eukaryotic and bacterial sequences are relatively low (in the 20% range), both the alignment of Fig. 3 and profile searches (data not shown) generated with either the Bacteria or the archaeal/eukaryotic family make clear their relatedness.

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Figure 3

Multiple sequence alignment of the eukaryotic translation initiation eIF-5A protein family with their archaeal homologs and the bacterial translation elongation EF-P family. The minimum domain of eukaryotic IF-5A needed for hypusine modification (51) is boxed; the asterisk denotes the lysine residue that is posttranslationally modified to hypusine. Protein names (and accession number—when different) are as follows: IF5A_METJA, M. jannaschii aIF-5A; IF5A-METTH, M. thermoautotrophicum unpublished ORF; IF5A-ARCFU, A. fulgidus unpublished ORF; IF5A_SULAC, Sulfolobus acidocaldarius aIF-5A; IF5A_HUMAN, human eIF-5A; IF5A_DICDI, Dictyostelium discoideum eIF-5A; IF51_YEAST, S. cerevisiae eIF-5A; IF52_CAEEL, Caenorhabditis elegans eIF-5A; EFP_BACSU, B. subtilis EF-P; EFP_SYNY3, Synechocystis sp. EF-P; EFP_ECOLI, E. coli EF-P; EFP_HELPY, Helicobacter pylori EF-P.

The archaeal IF-5A family displays an average of 32% and 26% identity to the eukaryotic and bacterial families, respectively, whereas bacterial and eukaryotes display an average of only 20%. The highest density of sequence conservation is found in the vicinity of the minimal region of the protein that must be conserved for lysine → hypusine conversion to occur (see boxed area of Fig. 3) (50).

Although hypusine is not present in the bacterial examples, a lysine residue usually is found in the corresponding position in the sequence. Note in Fig. 3 that in all archaeal and eukaryotic examples, the modified lysine residue is followed by histidine but that in all bacterial examples, an amino acid residue is absent at the corresponding position. Note also that bacterial EF-P sequences have a well-conserved C-terminal section that is absent in both archaeal and eukaryotic eIF-5A sequences, which suggests functional differentiation of the two molecular types.

A number of detailed suggestions have been made regarding eIF-5A function: the essential hypusine modification is suggested to stabilize Met-tRNA_i^Met binding to the peptidyltransferase center of the ribosome because formylation of the initiator Met residue (which removes its positive charge) renders it less dependent upon the presence of eIF-5A function (18). Complete intracellular depletion of eIF-5A (by gene deletion) results in inhibition of cell growth, although protein synthesis seems to be only slightly reduced (∼25%) (21). This has been interpreted to mean that eIF-5A may participate only in the translation of a subclass of mRNAs required directly for cell growth (21).