Characterization of the translational start site for IF2β, a short form of Escherichia coli initiation factor IF2

Abstract

The gene for initiation factor IF2, injB, represents one of the few examples in E.wherichiu coli of genes encoding two protein products in vim. In a previous work, our group showed that both forms of IF2 (a and p) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigourous determination of the nature of the initiation triplet for IF2p, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2a and IF2P. Three types of experiments were carried out. First, a 77-bp deletion was created at the beginning of the structural gene leading to premature termination of IF2a synthesis. Under these conditions, IF2p is still formed. Second, various Ba131 digests of infB containing the 77-bp deletion were fused to IucZ. Any synthesis of a fused protein with p-galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment. It was consequently possible to locate the IF2P initiation site within an 18-base region containing an in-phase GUG codon. Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lucZ an injB fragment devoid of IF2a start sequences but containing genetic information for this 18-base region. A hybrid protein with p-galactosidase activity was synthesized. Moreover, its NH2-terminal amino acid sequence coincided with that of IF2P, demonstrating that GUG, located 471 bases downstream from the IF2a external start codon, is the internal start codon for the shorter form of IF2.Initiation of translation in Escherichiu coli is a complex process that involves three protein factors IF1, IF2 and IF3. These initiation factors act either independently or jointly to catalyze the various reactions leading to the formation of the first peptide bond (for extensive reviews, see [l -31).IF2 interacts at least with GTP, met-tRNAr" and ribosomes to promote first the binding of Met-tRNAp' to 30 S ribosomal subunits and later to catalyse hydrolysis of GTP at the end of the initiation pathway [4]. As such, IF2 appears as an authentic soluble G protein sharing many common properties with other members of this protein family. Among the structural features of IF2 is the presence of a defined central G domain of 17 kDa, with an extensive sequence similarity with elongation factor Tu (EF-Tu) [5].IF2 can be isolated from cell lysates under three forms, named a, p and y [6], which differ in size (97.3 kDa, 79.7 kDa and 65.4 kDa respectively). Whereas IF27 is a truncated form resulting from proteolysis during purification [6], IF2a and IF2p appear to coexist in bacterial cells in a constant 2/1 molar ratio. It was initially shown that the a and P forms of IF2 are related immunologically and exhibit common tryptic peptides [7]. Amino acid sequence at the N-termini of the two forms as well as the overall amino acid sequence deduced from the nucleotide sequence of the IF2 gene, i...

Identification and Purification of Translation Initiation Factor 2 (IF2) from Thermus thermophilus

“…This indicates that the 66-kDa 7: thernzophilus IF2 is not a short form of the molecule formed by alternative initiation as observed in E. coli and in B. subtilis p forms of IF2 (Morel-Deville et al, 1990;Shazand et al, 1990). It also rules out the possibility that the 66-kDa polypeptide is a product of proteolytic degradation of a larger protein molecule, as was shown for the y form of E. coli elongation factor 2 (Cenatiempo et al, 1987).…”

“…Both forms of the protein are encoded by the in@ gene and are required for maximal cell growth (Sacerdot et al, 1992). The truncated p form results from an in-frame initiation event 158 codons downstream of the IF2a initiation codon (Plumbridge et al, 1985;Morel-Deville et al, 1990). Analysis of the IF2P initiation site revealed a second form of IF2P that initiates seven codons downstream of the internal 1F2/) initiation site (Sacerdot et al, 1992).…”

The Bradyrhizobium japonicum nolA Gene Encodes Three Functionally Distinct Proteins

“…In contrast, examples of multiple proteins derived from one prokaryotic gene have rarely been reported. There are a few examples (e.g., tipA, infB, clpA, clpB, and fbcH) where two prokaryotic polypeptides are derived from one gene (23,33,37,44,52). These products can be regulated translationally (e.g., clpA and clpB) via the use of alternative translational start sites on the same mRNA or posttranslationally via proteolytic processing (e.g., fbcH).…”

“…In a few cases, one gene has been shown to encode two proteins. Examples of these include tipA, infB, clpB, clpA, and fbcH (23,33,37,44,52). To our knowledge, there have been only two reports (for celA and PPI 3316 ) describing cases in which three proteins are encoded by one gene (3,34).…”

Translation initiation factor IF2 of the myxobacterium Stigmatella aurantiaca: presence of a single species with an unusual N-terminal sequence

“…IF2 is an essential protein in E. coli (23) and is present in the cell in two major cellular forms, ␣ (97.3 kDa) and ␤ (79.7 kDa) (27,28,33). It was also shown that IF2␤ comprises two barely distinguishable forms (␤1 and ␤2) arising from two internal in-frame initiation codons, GUG and AUG, respectively, separated by only 18 nucleotides (34).…”