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Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes
Published on: January 30, 2019
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Conserved small mRNA with an unique, extended Shine-Dalgarno sequence.
Julia Hahn1, Sebastian Thalmann1, Anzhela Migur1,2
1a Institute of Microbiology and Molecular Biology, Justus-Liebig-University , Gießen , Germany.
RNA Biology
|November 12, 2016
Summary
Researchers discovered a novel, small protein-coding gene, rreB, in Bradyrhizobiaceae. This gene exhibits unique ribosome binding site features, suggesting a new mechanism for gene regulation at the RNA level.
Area of Science:
- Microbiology
- Molecular Biology
- Genomics
Background:
- Very small protein-coding genes remain largely undiscovered in sequenced genomes.
- The identification and characterization of novel genes are crucial for understanding biological processes.
Purpose of the Study:
- To identify and characterize a novel, small mRNA and its encoded polypeptide in Bradyrhizobiaceae.
- To investigate the translational regulation and biophysical properties of the newly identified gene.
- To explore the potential for using unique ribosome binding site (RBS) features to discover similar genes in other bacterial genomes.
Main Methods:
- mRNA identification and sequencing.
- Localization in ribosome-containing fractions (P100).
- Co-sedimentation analysis with ribosomal subunits (sucrose density gradient).
- Translational fusion with egfp (enhanced green fluorescent protein).
- Biophysical characterization (CD- and NMR-spectroscopy).
- Bioinformatic analysis of bacterial genomes for conserved RBS sequences.
Main Results:
- A conserved 165-nucleotide mRNA encoding a 14-amino acid polypeptide (rreB) was identified in Bradyrhizobiaceae.
- The mRNA possesses a unique 17-nucleotide Shine-Dalgarno sequence, showing strong interaction with 30S ribosomal subunits.
- Translation was weak and impeded by the extended Shine-Dalgarno sequence and a GTG start codon.
- The encoded polypeptide was unstructured, and transcript stability increased when the start codon was replaced by a stop codon, indicating post-transcriptional regulation.
- Homologous genes (rreR) with similar RBS features were found in other α-proteobacteria, such as Caulobacterales, Rhizobiales, and Rhodobacterales.
Conclusions:
- The study identified a novel small gene, rreB, regulated at the ribosome level.
- Unique RBS features can serve as a marker for discovering similarly regulated genes in bacteria.
- This work expands our understanding of gene regulation mechanisms in prokaryotes.
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