Two translational initiation sites in the infB gene are used to express initiation factor IF2 alpha and IF2 beta in Escherichia coli.

Abstract

The gene infB codes for the two forms of translational initiation factor IF2: IF2 alpha (97 300 daltons) and IF2 beta (79 700 daltons). To determine whether the two forms differ at their N terminus, purified IF2 alpha and IF2 beta were subjected to 11 or more steps of Edman degradation. The N‐terminal amino acid sequences are completely different, but match perfectly the DNA sequences at the beginning of the infB open reading frame and an in‐phase region 471 bp downstream. A fusion was constructed between the proximal half of the infB gene and the lacZ gene lacking the region coding for the first eight amino acids. The fused gene expresses two products of 170 000 and 150 000 daltons, corresponding to the fused proteins IF2 alpha‐beta‐galactosidase and IF2 beta‐beta‐galactosidase, which confirms in vivo that the IF2 forms differ at their N terminus. A deletion of the 5′‐non‐translated region of the fused gene, including the Shine/Dalgarno ribosomal binding site, results in the expression of IF2 beta‐beta‐galactosidase but not IF2 alpha‐beta‐galactosidase. This strongly suggests that IF2 beta results from independent translation rather than from a precise proteolytic cleavage of IF2 alpha. Further evidence for initiation of protein synthesis at the putative IF2 alpha and IF2 beta start sites was sought by using an in vitro dipeptide synthesis assay. A DNA fragment containing the entire infB gene was cloned into three plasmid vectors and the resulting recombinant DNAs were used as templates in assays containing fMet‐tRNA and various labelled aminoacyl‐tRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…In contrast, no obvious ribosome binding site for I F 2 j synthesis could be easily detected within the structural infB gene. However, in a previous report [8], several results strongly supported the existence of two independent translational start sites, used to express the two forms of initiation factor IF2. This assertion was based on an in vivo synthesis of IF2P-P-galactosidase hybrid protein after deletion of the upstream region, including the Shine and Dalgarno sequence, in front of the AUG codon for IF2a and strengthened by in vitro experiments using a dipeptide assay system [16, 171. In the latter assay two W e t dipeptides were obtained from the cloned gene, one of which suggested the position and the nature of the internal start site for IF2P synthesis.…”

“…When subcloned as a 3-kbp segment, infB expresses both forms of IF2, found in a constant ratio either in exponentially growing cells, in maxicell or minicell systems [8,111 (and F. Morel-Deville, unpublished results). S1 nuclease mapping experiments indicate that all the genes are co-transcribed from the primary promoter located upstream of met Y .…”

“…Avenue du Recteur Pineau, F-86022 Poitiers Cedex, France examined [8,91. It was found that IF28 is precisely identical to 80% of IF2a from the C-terminus, but lacks 157 amino acids of the N-terminus.…”

“…S1 nuclease mapping experiments indicate that all the genes are co-transcribed from the primary promoter located upstream of met Y . Under some circumstances, the activity of internal promoters, particularly for IF2, could be observed [8,15], but their role in vivo remains questionable. It is still believed that IF2a as well as IF2p arise from the longest reading frame (2667 bases) in infB, i.e.…”

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

“…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).…”