IF2 Alpha Research Articles

Protein signatures (molecular synapomorphies) that are distinctive characteristics of the major cyanobacterial clades

A combination of phylogenomic and signature sequence-based (or phenetic) approaches was used to understand the evolutionary relationships among cyanobacteria. Phylogenetic trees were constructed for 34 cyanobacteria whose genomes have been sequenced, based on concatenated sequences for 45 conserved proteins and also the 16S rRNA gene. In parallel, sequence alignments of various proteins were examined to identify conserved indels (i.e. molecular signatures or synapomorphies) that are specific for either all cyanobacteria or their various clades in the phylogenetic trees. Of the >40 molecular signatures described in this work, 15 are specific for all cyanobacteria. The other cyanobacterial clades that can now be identified and circumscribed in molecular terms by using these signatures include a deep-branching clade (clade A, corresponding to the subclass Gloeobacterophycidae), consisting of Gloeobacter violaceus and two diazotrophic Synechococcus strains (JA-3-3Ab and JA2-3-B'a) (15 aa insert in EF-G); a clade comprising all other cyanobacteria except those from clade A [18 aa insert in DNA polymerase I (Pol I), 2 aa insert in the DnaX protein, 4 aa insert in TrpRS and 4-5 aa insert in tryptophan synthase beta subunit]; a clade (clade C, corresponding to the subclass Synechococcophycidae) of various marine unicellular Synechococcus and Prochlorococcus cyanobacteria (12 aa insert in Pol I, 3 aa insert in RpoB, 2 aa insert in KgsA, 6 aa insert in TyrRS, 2 aa insert in tRNA-mG1 transferase and 1 aa deletion in the RpoC protein); a clade of the low-B/A ecotype Prochlorococcus strains (5 aa deletion in LeuRS and 1 aa insert in the Ffh protein); a clade consisting of the Nostocales species/strains (subclass Nostocophycidae; 4 aa insert in the PetA protein and 5 aa insert in the ribosomal protein S3); a clade of the order Chroococcales (1 aa insert in RecA); a clade comprising the orders Nostocales, Oscillatoriales and Chroococcales [19 aa insert in DnaE, 13 aa insert in GDP-mannose pyrophosphorylase and 22-27 aa insert in NADP(H)-quinone oxidoreductase subunit D]. Two additional conserved indels in the translation-initiation factor IF-2 and riboflavin synthase alpha subunit suggest an intermediate placement of the Oscillatoriales in between the orders Nostocales and Chroococcales. The unique presence of these molecular signatures in all available sequences from the indicated groups of cyanobacteria, but not in any other cyanobacteria (or bacteria), indicates that these synapomorphies provide novel and potentially useful means for circumscription of several important taxonomic clades of cyanobacteria in more definitive terms. The species-distribution patterns of these synapomorphies also indicate that the plant/plastid homologues are not derived from the clade A or C cyanobacteria.

Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2

Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2 alpha (IF2 alpha). Subsequent Southern blot analysis of B. subtilis genomic DNA identified a 1.3-kilobase (kb) HindIII fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindIII fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The HindIII fragment was also used as a probe to obtain overlapping clones from a lambda gt11 library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis infB gene encodes two proteins, IF2 alpha (78.6 kilodaltons) and IF2 beta (68.2 kilodaltons); both were expressed in B. subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2 alpha and were able to complement in vivo an E. coli infB gene disruption. Four-factor recombination analysis positioned the infB gene at 145 degrees on the B. subtilis chromosome, between the polC and spcB loci. This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.

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

The gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo. In a previous work, our group showed that both forms of IF2 (alpha and beta) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigorous determination of the nature of the initiation triplet for IF2 beta, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2 alpha and IF2 beta. 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 IF2 alpha synthesis. Under these conditions, IF2 beta is still formed. Second, various Bal31 digests of infB containing the 77-bp deletion were fused to lacZ. Any synthesis of a fused protein with beta-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 IF2 beta 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 lacZ an infB fragment devoid of IF2 alpha start sequences but containing genetic information for this 18-base region. A hybrid protein with beta-galactosidase activity was synthesized. Moreover, its NH2-terminal amino acid sequence coincided with that of IF2 beta, demonstrating that GUG, located 471 bases downstream from the IF2 alpha external start codon, is the internal start codon for the shorter form of IF2.

In vitro expression of the Escherichia coli nusA-infB operon.

The expression of the nusA-infB operon has been investigated using an in vitro system based on the formation of the first dipeptide of the gene product. A series of plasmids containing various deletions of the operon were used as templates in this study. Of the four genes coding for protein products, 15Ka, nusA, infB, and 15Kb, only 15Ka was not expressed in this dipeptide system. The initial dipeptides for the other gene products, fMet-Asn (pnusA), fMet-Thr (IF-2 alpha), and fMet-Ala (p15Kb), were synthesized even from plasmids lacking the primary promoters. It appears that secondary (internal) promoters in the operon can efficiently direct the expression of these genes. No regulation of the expression was observed with IF-2 alpha, but pnusA inhibited the expression of the nusA gene (autoregulation) as well as the p15b gene. Experiments using an uncoupled system indicated that the effect of pnusA on nusA expression was at the level of transcription, but that both a transcriptional and a post-transcriptional effect of pnusA was seen on 15Kb expression.

Overproduction and purification of initiation factor IF2 and pNUSA proteins from a recombinant plasmid bearing strain

The genes for translational initiation factor, IF2 and pNusA have been cloned into a plasmid vector where they are placed under the control of the inducible lambdapL promoter and the c1857 thermosensitive repressor. When a strain carrying this plasmid is heat induced, IF2 alpha, IF2 beta and pNusA are overproduced 15 to 20 fold. This has allowed us to purify the IF2 and NusA proteins in large amounts.

In vivo evidence that the nusA and infB genes of E. coli are part of the same multi-gene operon which encodes at least four proteins.

Previous work has shown that the Escherichia coli nusA gene codes for a protein which regulates transcription termination. The 16.0-kb EcoRI DNA fragment that includes the nusA gene, codes for at least eight bacterial proteins of mol. wts. 48 000 (argG), 21 000 (p21), 64 000 (nusA), 120 000 (IF2 alpha)-(infB), 91 000 (IF2 beta)(infB), 15 000 (p15), 10 000 (rpsO) and 85 000 (pnp). We have constructed several deletion and fusion derivatives from this cloned DNA and examined in vivo the structure and expression of these genes. First, the promoter functional in vivo for the nusA gene was mapped at approximately 800 bp upstream of the nusA structural gene. Second, the synthesis of five proteins, p21, NusA, IF2 alpha, IF2 beta (and p15) proteins, was affected by the deletion of the nusA promoter. Third, these same five proteins were hyperproduced after fusion of the DNA fragment to the lambda pL promoter. In addition, subcloning experiments revealed that the p15 gene is expressed by the read-through transcription from the infB gene. These results lead us to conclude that the genes coding for the p21, NusA, InfB (IF2 alpha and IF2 beta), and p15 proteins form a single-transcriptional unit ('nusA-infB operon') in vivo and that rpsO and pnp genes do not belong to the same operon. The in vivo attenuation site of this operon is described.

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

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)

The nucleotide sequence of the cloned nusA gene and its flanking region of Escherichia coli.

The nucleotide sequence of the promoter-proximal portion of the nusA operon including the genes for tRNAMetf2, a 15 kilodalton protein and the initial portion of the nusA gene has been determined previously (1). Here, we report the sequence for the entire nusA gene and its flanking region. The open reading frame, consisting of 1,482 nucleotides, was identified as that of the nusA protein on the basis of agreement of the amino acid sequence deduced from the DNA sequence with the N-terminal sequence of the purified nusA protein. The molecular weight of 54,417 daltons calculated for the 494 amino acid polypeptide is significantly lower than that determined previously by SDS polyacrylamide gel analysis. The nusA gene is immediately followed by another open reading frame encoding a polypeptide of at least 22 amino acids, which was identified as the initial portion of the infB structural gene. In the spacer region of 24 base pairs between the nusA and infB structural genes there is no significant DNA sequence that fits the canonical transcriptional termination signal or promoter sequence. We suggest, therefore, that the genes for tRNAMetf2, a 15 kilodalton protein, the nusA protein and IF2 alpha, aligned in this order, are co-transcribed.