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

Abstract

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 α-proteobacteria 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.

The Leader Peptide peTrpL Forms Antibiotic-Containing Ribonucleoprotein Complexes for Posttranscriptional Regulation of Multiresistance Genes

“…Plasmid pSUP202pol4 (60) was used for construction of an integration vector for S. meliloti. Because of the lack of a suitable inducible plasmid for B. japonicum, for peptide overproduction in this organism, the constitutive promoter-containing integration vector pRJPaph-MCS was used (65).…”

Rapid Biophysical Characterization and NMR Spectroscopy Structural Analysis of Small Proteins from Bacteria and Archaea

“…We have established a routine work protocol and screened 27 small proteins for secondary structure. Within this screening, we observed all possible conformations: 1) entirely unstructured proteins (random‐coil state): 2) proteins with a fluctuating amount of residual structure, but no defined tertiary fold (molten globule), as well as specific structures with only lowly populated excited states; and 3) structured proteins . We report herein on the protocol for structure determination of one exemplary small protein (SP‐22; PDB ID: 6Q2Z; BMRB ID: 34334) .…”

Transcription attenuation-derived small RNA rnTrpL regulates tryptophan biosynthesis gene expression in trans

“…In S. meliloti , pRK-rnTrpL leads to constitutive transcription of rnTrpL from a heterologous ribosomal RNA ( rrn ) promoter originating from the alpha-proteobacterium Rhodobacter shpaeroides (41). For constitutive overexpression of the B. japonicum rnTrpL homolog Bja-rnTrpL, the corresponding sequence was cloned in the chromosome integration plasmid pRJ-MCS (44) that was cleaved with KpnI and SpeI. Plasmid pRK-rnTrpL-AU1,2UA, which allows for constitutive transcription of an S. meliloti rnTrpL derivative that lacks a functional sORF (replacement of the trpL AUG codon with UAG), was constructed as follows.…”