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RNA Biol. 2017 Feb;14(2):206-218. doi: 10.1080/15476286.2016.1270001. Epub 2016 Dec 16.

Systematic characterization of artificial small RNA-mediated inhibition of Escherichia coli growth.

Author information

1
a Institute for Advanced Biosciences, Keio University , Tsuruoka , Japan.
2
b Systems Biology Program, Graduate School of Media and Governance, Keio University , Fujisawa , Japan.
3
c Faculty of Environment and Information Studies, Keio University , Fujisawa , Japan.

Abstract

A new screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli was constructed. In this system, we used a plasmid library to express RNAs of ∼120 nucleotides, each with a random 30-nucleotide sequence that can recognize its target mRNA(s). After approximately 60,000 independent colonies were screened, several plasmids that inhibited bacterial growth were isolated. To understand the inhibitory mechanism, we focused on one sRNA, S-20, that exerted a strong inhibitory effect. A time-course analysis of the proteome of S-20-expressing E. coli and a bioinformatic analysis were used to identify potential S-20 target mRNAs, and suggested that S-20 binds the translation initiation sites of several mRNAs encoding enzymes such as peroxiredoxin (osmC), glycyl-tRNA synthetase α subunit (glyQ), uncharacterized protein ygiM, and tryptophan synthase β chain (trpB). An in vitro translation analysis of chimeric luciferase-encoding mRNAs, each containing a potential S-20 target sequence, indicated that the translation of these mRNAs was inhibited in the presence of S-20. A gel shift analysis combined with the analysis of a series of S-20 mutants suggested that S-20 targets multiple mRNAs that are responsible for inhibiting E. coli growth. These data also suggest that S-20 acts like an endogenous sRNA and that E. coli can utilize artificial sRNAs.

KEYWORDS:

Artificial small RNA; Escherichia coli; RNA-RNA interaction; growth inhibition; proteomic analysis; reporter assay; target mRNA

PMID:
27981881
PMCID:
PMC5324740
DOI:
10.1080/15476286.2016.1270001
[Indexed for MEDLINE]
Free PMC Article

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