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 Bradyrhizobiaceae 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 Bradyrhizobium japonicum 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 egfp, 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 rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB 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 Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of Alphaproteobacteria the corresponding gene (rreR) 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.迄今为止，测序基因组中仍有大量微小蛋白编码基因尚未被发掘鉴定。本研究鉴定出一条长度为165个核苷酸（nucleotides，nt）的mRNA，该序列在慢生根瘤菌科（Bradyrhizobiaceae）中具有进化保守性，其编码一条含14个氨基酸残基（amino acid residues，aa）的多肽。该小型mRNA携带一段长度为17 nt的独特夏因-达尔加诺序列（Shine-Dalgarno sequence，SD），主要定位于大豆慢生根瘤菌（Bradyrhizobium japonicum）USDA 110的含核糖体P100组分中。通过蔗糖密度梯度共沉降实验，证实该mRNA与30S核糖体亚基存在强相互作用。借助与增强绿色荧光蛋白（enhanced green fluorescent protein，egfp）的翻译融合实验，我们检测到了微弱的翻译活性，并发现该翻译过程会被延长的SD序列以及GTG起始密码子（而非常规的ATG起始密码子）所阻碍。生物物理表征（圆二色谱（CD）与核磁共振波谱（NMR））结果显示，合成的多肽在生理缓冲液中仍呈无结构状态。将起始密码子替换为终止密码子可提高该转录本的稳定性，这强烈提示该基因在核糖体层面存在额外的转录后调控机制。因此，我们将该小型基因命名为rreB（慢生根瘤菌科核糖体调控表达基因，全称ribosome-regulated expression in Bradyrhizobiaceae）。鉴于该独特的核糖体结合位点（ribosome binding site，RBS）可作为rreB同源基因或受类似调控的基因的标志性序列，我们在细菌基因组中搜寻了类似的推定RBS，并在柄杆菌目（Caulobacterales）、根瘤菌目（Rhizobiales）、红杆菌目（Rhodobacterales）以及红螺菌目（Rhodospirillales）的小型开放阅读框（small open reading frames，sORFs）上游区域，检测到了与16S rRNA 3'端存在至少16 nt互补性的区域。在α-变形菌纲的红杆菌/玫瑰杆菌分支中，对应的基因（rreR）具有进化保守性，其编码一条含18个氨基酸残基的蛋白质。本研究证实，可通过特定的RBS特征来鉴定那些推测在RNA层面受类似表达调控的新基因。