Unidirectional gene pairs in archaea and bacteria require overlaps or very short intergenic distances for translational coupling via termination-reinitiation and often encode subunits of heteromeric complexes.

Genomes of bacteria and archaea contain a much larger fraction of unidirectional (serial) gene pairs than convergent or divergent gene pairs. Many of the unidirectional gene pairs have short overlaps of -4 nt and -1 nt. As shown previously, translation of the genes in overlapping unidirectional gene pairs is tightly coupled. Two alternative models for the fate of the post-termination ribosome predict either that overlaps or very short intergenic distances are essential for translational coupling or that the undissociated post-termination ribosome can scan through long intergenic regions, up to hundreds of nucleotides. We aimed to experimentally resolve the contradiction between the two models by analyzing three native gene pairs from the model archaeon Haloferax volcanii and three native pairs from Escherichia coli. A two reporter gene system was used to quantify the reinitiation frequency, and several stop codons in the upstream gene were introduced to increase the intergenic distances. For all six gene pairs from two species, an extremely strong dependence of the reinitiation efficiency on the intergenic distance was unequivocally demonstrated, such that even short intergenic distances of about 20 nt almost completely abolished translational coupling. Bioinformatic analysis of the intergenic distances in all unidirectional gene pairs in the genomes of H. volcanii and E. coli and in 1,695 prokaryotic species representative of 49 phyla showed that intergenic distances of -4 nt or -1 nt (= short gene overlaps of 4 nt or 1 nt) were by far most common in all these groups of archaea and bacteria. A small set of genes in E. coli, but not in H. volcanii, had intergenic distances of around +10 nt. Our experimental and bioinformatic analyses clearly show that translational coupling requires short gene overlaps, whereas scanning of intergenic regions by the post-termination ribosome occurs rarely, if at all. Short overlaps are enriched among genes that encode subunits of heteromeric complexes, and co-translational complex formation requiring precise subunit stoichiometry likely confers an evolutionary advantage that drove the formation and conservation of overlapping gene pairs during evolution.

Translational coupling via termination-reinitiation in archaea and bacteria.

The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria.

Non-canonical Escherichia coli transcripts lacking a Shine-Dalgarno motif have very different translational efficiencies and do not form a coherent group.

Translation initiation in 50-70 % of transcripts in Escherichia coli requires base pairing between the Shine-Dalgarno (SD) motif in the mRNA and the anti-SD motif at the 3' end of the 16S rRNA. However, 30-50 % of E. coli transcripts are non-canonical and are not preceded by an SD motif. The 5' ends of 44 E. coli transcripts were determined, all of which contained a 5'-UTR (no leaderless transcripts), but only a minority contained an SD motif. The 5'-UTR lengths were compared with those listed in RegulonDB and reported in previous publications, and the identities and differences were obtained in all possible combinations. We aimed to quantify the translational efficiencies of non-canonical 5'-UTRs using GusA reporter gene assays and Northern blot analyses. Ten non-canonical 5'-UTRs and two control 5'-UTRs with an SD motif were cloned upstream of the gusA gene. The translational efficiencies were quantified under five different conditions (different growth rates via two different temperatures and two different carbon sources, and heat shock). The translational efficiencies of the non-canonical 5'-UTRs varied widely, from 5 to 384 % of the positive control. In addition, the non-canonical transcripts did not exhibit a common regulatory pattern with changing environmental parameters. No correlation could be observed between the translational efficiencies of the non-canonical 5'-UTRs and their lengths, sequences, GC content, or predicted secondary structures. The introduction of an SD motif enhanced the translational efficiency of a poorly translated non-canonical transcript, while the efficiency of a well-translated non-canonical transcript remained unchanged. Taken together, the mechanisms of translation initiation at non-canonical transcripts in E. coli still need to be elucidated.

The glpD gene is a novel reporter gene for E. coli that is superior to established reporter genes like lacZ and gusA.

Reporter genes facilitate the characterization of promoter activities, transcript stabilities, translational efficiencies, or intracellular localization. Various reporter genes for Escherichia coli have been established, however, most of them have drawbacks like transcript instability or the inability to be used in genetic selections. Therefore, the glpD gene encoding glycerol-3-phosphate dehydrogenase was introduced as a novel reporter gene for E. coli. The enzymatic assay was optimized, and it was verified that growth on glycerol strictly depends on the presence of GlpD. The 5'-UTRs of three E. coli genes were chosen and cloned upstream of the new reporter gene glpD as well as the established reporter genes lacZ and gusA. Protein and transcript levels were quantified and translational efficiencies were calculated. The lacZ transcript was very unstable and its level highly depended on its translation, compromising its use as a reporter. The results obtained with gusA and glpD were similar, however, only glpD can be used for genetic selections. Therefore, glpD was found to be a superior novel reporter gene compared to the established reporter genes lacZ and gusA.