ИСТИНА

A great variety of small non-coding RNAs are known to play different regulatory roles in bacteria. While mRNAs quite rarely are involved in any regulation and mostly act as templates for protein synthesis. In our previous research, we identified an RNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and harbor a unique Shine-Dalgarno sequence (SD) with a length of 17 nt. This RNA is weakly synthesized under different growth conditions in B. japonicum USDA 110, but strongly interacts with 16S rRNA of 30S ribosomal subunits showing ability to be a potential translational inhibitor. Therefore, its coding gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). On the other hand, RreB RNA possess small ORF that encodes a polypeptide with 14 amino acid residues, which miserable translation was shown using fusions with egfp. Thus, it’s unclear up now whether RreB RNA displays its function as non-coding regulatory RNA and inhibit ribosome or it could be translated and gives rise to a small peptide with unknown function. Finally, it could possess a double function. RreB homologs also were predicted in other families of alpha-proteobacteria. Interestingly, four putative candidates with 10-14 nt SD followed by small ORFs were found in S. meliloti. In the present research, we aimed identification of those RNAs by RT-PCR and Northern blotting as well as verification of their ability to bind 30S subunit and serve as templates for synthesis of the corresponding peptides. To check translation activity of those RNAs, we substitute ORFs by mCherry gene saving original 5’-UTRs and clone constructs in a vector under control of a strong ribosomal promoter. We also obtained vectors for protein super expression where substitute original 5’-UTRs with a canonical one and put the construct under IPTG control. This work was supported by the Russian Scientific Foundation (grant N14-24-00061).