Conserved small mRNA with an unique, extended Shine-Dalgarno sequence

Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in <i>Bradyrhizobiaceae</i> and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of <i>Bradyrhizobium japonicum</i> USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with <i>egfp</i>, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named <i>rreB</i> (ribosome-regulated expression in <i>Bradyrhizobiaceae</i>). Assuming that the unique ribosome binding site (RBS) is a hallmark of <i>rreB</i> homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in <i>Caulobacterales, Rhizobiales, Rhodobacterales</i> and <i>Rhodospirillales</i>. In the <i>Rhodobacter/Roseobacter</i> lineage of Alphaproteobacteria the corresponding gene (<i>rreR</i>) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level.