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Hfq-dependent regulation of OmpA synthesis is mediated by an an-tisense RNA.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2005 (English)In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 19, no 19, 2355-2366 p.Article in journal (Refereed) Published
Abstract [en]

This paper shows that the small RNA MicA (previously SraD) is an antisense regulator of ompA in Escherichia coli. MicA accumulates upon entry into stationary phase and down-regulates the level of ompA mRNA. Regulation of ompA (outer membrane protein A), previously attributed to Hfq/mRNA binding, is lost upon deletion of the micA gene, whereas overexpression of MicA inhibits the synthesis of OmpA. In vitro, MicA binds to the ompA mRNA leader. Enzymatic and chemical probing was used to map the structures of MicA, the ompA mRNA leader, and the complex formed upon binding. MicA binding generates a footprint across the ompA Shine-Dalgarno sequence, consistent with a 12 + 4 base-pair interaction, which is additionally supported by the effect of mutations in vivo and by bioinformatics analysis of enterobacterial micA/ompA homolog sequences. MicA is conserved in many enterobacteria, as is its ompA target site. In vitro toeprinting confirmed that binding of MicA specifically interferes with ribosome binding. We propose that MicA, when present at high levels, blocks ribosome binding at the ompA translation start site, which—in line with previous work—secondarily facilitates RNase E cleavage and subsequent mRNA decay. MicA requires the presence of the Hfq protein, although the mechanistic basis for this remains unclear.

Place, publisher, year, edition, pages
2005. Vol. 19, no 19, 2355-2366 p.
Keyword [en]
Antisense RNA, Hfq, OmpA, regulatory RNA, translational control
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-95610DOI: 10.1101/gad.354405OAI: oai:DiVA.org:uu-95610DiVA: diva2:169899
Available from: 2007-03-23 Created: 2007-03-23 Last updated: 2014-05-28Bibliographically approved
In thesis
1. Functional characterization of the small antisense RNA MicA in Escherichia coli
Open this publication in new window or tab >>Functional characterization of the small antisense RNA MicA in Escherichia coli
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Escherichia coli small RNA (sRNA) MicA was identified recently in a genomewide search for sRNAs. It is encoded between the genes gshA and luxS in E. coli and its close relatives. The function of sRNAs in bacteria is generally believed to be in maintenance of homeostasis via stress-induced modulation of gene expression. Our studies on MicA have been aimed at attributing function(s) to this molecule.

We carried out high throughput assays aimed at identifying genes that are differentially regulated upon knocking out or overexpressing MicA. Among the protein candidates identified was the outer membrane protein, OmpA. Subsequent analysis allowed us to show this regulation to be antisense in nature with MicA binding within the translation initiation region of ompA mRNA. Furthermore, blocking the ribosome from loading caused a translational decoupling that instigates degradation of the mRNA. The regulation was apparent in early stationary phase and seen to be dependent on the RNA chaperone Hfq.

We went on to characterize the regulation of MicA, looking at its own transcription. Testing various stress conditions, we were able to identify putative promoter elements that we confirmed using transcriptional fusions. The results showed MicA to be dependent on the extracytoplasmic function ECF sigma E (σE) and could not detect MicA in mutants deleted for this factor.

Lastly, we identified an additional target for MicA being the adjacently encoded luxS mRNA. The LuxS protein is essential for the synthesis of the quorum sensing AI-2 molecule. Transcription of the luxS mRNA is commences within the gshA gene, on the other side of MicA coding region. We were able to show that MicA interacts with luxS mRNA and is recognized by RNase III which processes this complex leading to a shorter luxS mRNA isoform. The significance of this processing event is as yet undetermined. Our data elucidated a new promoter driving transcription of luxS, and we demonstrated this promoter to be stationary phase responsive.

In summary, the work presented here characterizes the sRNA MicA as a dual regulatory sRNA molecule, moonlighting between its cis-encoded target and its trans-encoded target. .

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 67 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 284
small RNA, antisense, outer membrane, Hfq-dependence, RNase III, LuxS, Quorum sensing
National Category
Microbiology in the medical area
Research subject
urn:nbn:se:uu:diva-7759 (URN)978-91-554-6833-0 (ISBN)
Public defence
2007-04-13, B10:1, BMC, Husargatan 3, Uppsala, 09:00
Available from: 2007-03-23 Created: 2007-03-23Bibliographically approved

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Holmqvist, ErikWagner, Gerhart. E. H.
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