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Two antisense RNAs target the transcriptional regulator CsgD to inhibit curli synthesis
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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2010 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 29, no 11, p. 1840-1850Article in journal (Refereed) Published
Abstract [en]

Escherichia coli produces proteinaceous surface structures called curli that are involved in adhesion and biofilm formation. CsgD is the transcriptional activator of curli genes. We show here that csgD expression is, in part, controlled post-transcriptionally by two redundant small RNAs (sRNAs), OmrA and OmrB. Their overexpression results in curli deficiency, in accordance with the inhibition of chromosomally encoded, FLAG-tagged CsgD. Downregulation of csgD occurs by a direct antisense interaction within the csgD 5'-UTR, far upstream of the ribosome-binding site (RBS). OmrA/B downregulate plasmid-borne csgD-gfp fusions in vivo, and inhibit CsgD translation in vitro. The RNA chaperone Hfq is required for normal csgD mRNA and OmrA/B levels in the cell, and enhances sRNA-dependent inhibition of csgD translation in vitro. Translational inhibition involves two phylogenetically conserved secondary structure modules that are supported by chemical and enzymatic probing. The 5'-most element is necessary and sufficient for regulation, the one downstream comprises the RBS and affects translational efficiency. OmrA/B are two antisense RNAs that regulate a transcription factor to alter a morphotype and group behaviour.

Place, publisher, year, edition, pages
2010. Vol. 29, no 11, p. 1840-1850
Keyword [en]
antisense RNA, bacterial sRNA, curli regulation, Hfq, transcriptional activator
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-131164DOI: 10.1038/emboj.2010.73ISI: 000278235100007PubMedID: 20407422OAI: oai:DiVA.org:uu-131164DiVA, id: diva2:353363
Available from: 2010-09-26 Created: 2010-09-26 Last updated: 2018-02-25Bibliographically approved
In thesis
1. Macromolecular Matchmaking: Mechanisms and Biology of Bacterial Small RNAs
Open this publication in new window or tab >>Macromolecular Matchmaking: Mechanisms and Biology of Bacterial Small RNAs
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability.

By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. p. 66
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 916
Keyword
Small RNA, sRNA, non-coding RNA, antisense, gene regulation, post-transcriptional regulation, translational control, outer membrane protein, OmpA, MicA, biofilm, flagella, curli, bistability, Lrp, MicF, CsgD, FlgM, OmrA, OmrB
National Category
Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-171642 (URN)978-91-554-8331-9 (ISBN)
Public defence
2012-05-16, Sal B42, Biomedicinskt Centrum, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-04-24 Created: 2012-03-25 Last updated: 2012-08-01Bibliographically approved
2. Ribosome standby sites and other structural aspects of translation initiation regions in Escherichia coli
Open this publication in new window or tab >>Ribosome standby sites and other structural aspects of translation initiation regions in Escherichia coli
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Translation initiation, which is rate-limiting in protein synthesis, is often the step at which regulation occurs. Here, we investigated several mechanisms of translation initiation in Escherichia coli, including their control. First, we showed that translation of the transcriptional regulator CsgD is inhibited by two sRNAs through a direct antisense mechanism.

In some bacterial mRNAs, the ribosome binding site (RBS) is sequestered in a stable structure, which generally generates very low protein output. Yet, these mRNAs are often efficiently translated, which suggested the requirement for “ribosome standby sites”. Here, we investigated the structure and sequence features of an effective standby site using plasmid-borne GFP reporter constructs, and showed that relatively short, single-stranded regions near a structurally sequestered RBS can profoundly increase translation rates. Both the length and the sequence of these single-stranded regions are important for standby site efficiency, and the standby site needs to be single-stranded. This work serves as a proof-of-principle study of the ribosome standby model.

To investigate the sequence-dependency of standby sites further, we used an unbiased approach, creating plasmid libraries containing millions of different standby sites in the same reporter plasmid as before. Cells were sorted by fluorescence according to translational levels, and standby sites analyzed by deep sequencing. This analysis showed that efficient standby sites have a low GC-content and rarely contain Shine-Dalgarno sequences. Additionally, nucleotides near the 3’-border of the standby region affect translation efficiency more than those closer to the 5’-end. Mutational and structure-probing experiments are planned to verify these findings.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 58
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1636
Keyword
Ribosome standby sites, translation initiation, mRNA structure, bacteria
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-343082 (URN)978-91-513-0249-2 (ISBN)
Public defence
2018-04-13, Room A1:111a, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2018-03-23 Created: 2018-02-25 Last updated: 2018-04-24

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