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The first 20 codons of the prfA-mRNA are required for efficient translation in Listeria monocytogenes
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). (Jörgen Johansson)
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Berit Sondén)
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). (Jörgen Johansson)
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Berit Sondén)
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Expression of virulence factors in the human pathogen Listeria monocytogenes is almost exclusively regulated by the transcriptional activator PrfA. The translation of prfA is controlled by a thermosensor located in the 5´-untranslated RNA (UTR), which is high at 37°C and low at temperatures below 30°C. Also, translation of the prfA transcript is inhibited by a trans-acting riboswitch RNA, SreA, which binds to the 5´-end of the thermosensor. In order to develop a thermoregulated translational expression system in Mycobacterial species, the 5´-UTR and different lengths of the prfA-coding sequences were placed in front of lacZ. When expressed in Escherichia coli, the constructs retained their thermoregulation. However, the β-galactosidase expression was directly correlated to the length of the prfA-coding mRNA fused in front of lacZ. A similar regulation was also detected when gfp was used as a reporter gene. Transcriptional stability experiments indicated that the observed difference in expression was not due to a decreased stability of transcripts lacking more of the prfA-coding RNA. The gfp constructs behaved similarly in L. monocytogenes as in E. coli, emphasizing the requirement of the prfA-coding RNA for maximal expression, also in its natural genetic background. Moreover, the different PrfA-LacZ fusion proteins showed the same proteolytic stability, ruling out post-translational mechanisms. Instead, in vitro transcription/translation experiments suggest a role of the first 20 codons of the native prfA-mRNA for maximal expression. Our data indicated that the difference in expression was not due to rare codons, stretches of certain bases or a putative downstream box. We observed an inverse correlation between the stability of the RNA secondary structure and protein expression. The first 12 codons of prfA displayed a very weak RNA secondary structure. Similar weak stabilities were detected also for thermosensors in other species, indicating a common strategy of regulation. In summary, the present work determines the importance of an unstructured 5´-coding region of the prfA-RNA for efficient translation.

Keyword [en]
Listeria, prfA-mRNA
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-33103OAI: oai:DiVA.org:umu-33103DiVA: diva2:310097
Available from: 2010-04-12 Created: 2010-04-12 Last updated: 2010-04-20Bibliographically approved
In thesis
1. RNA-mediated virulence gene regulation in the human pathogen Listeria monocytogenes
Open this publication in new window or tab >>RNA-mediated virulence gene regulation in the human pathogen Listeria monocytogenes
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Gram-positive human pathogen Listeria monocytogenes uses a wide range of virulence factors for its pathogenesis. The majority of its virulence genes are encoded on a 9-kb pathogenicity island and are controlled by the transcriptional activator PrfA. Expression of these genes is maximal at 37°C and minimal at 30°C in a mechanism involving an RNA thermosensor. This thesis brings up different aspects of RNA-mediated regulation, including regulatory RNA structures within coding mRNA controlling expression to 5-untranslated RNA (5´-UTR) that controls downstream genes (cis-acting) as well as small non-coding RNAs (ncRNAs) that bind other target RNA (trans-acting).

We investigated the importance of the coding region of the prfA-mRNA for its expression. Various lengths of prfA-mRNA were fused with reporter genes. Our finding suggested that the first 20 codons of prfA-mRNA were essential for efficient translation in Listeria monocytogenes. Translation of the shorter constructs was shown to be reduced. The expression level showed an inverse correlation with the RNA secondary structure stability in the beginning of the coding region. Riboswitches have previously been known to control expression of their downstream mRNA in a cis-acting manner. A trans-acting S-adenosylmethionine-binding riboswitch termed SreA was identified in Listeria monocytogenes. It was found to control the expression of the virulence regulator PrfA, by binding to the prfA-UTR and thereby affecting its translation. We examined the RNA locus encoding different virulence factors in Listeria monocytogenes. Several of them were preceded by 5´-UTRs of various lengths. We speculate that these 5´-UTRs could control expression of the downstream mRNA, provided they are of sufficient length. These findings prompted us to examine where and when Listeria monocytogenes switches on gene expression. Tiling array was used to compare RNAs isolated from wild-type and mutant bacteria grown at different growth conditions. Antisense RNAs covering parts of or whole open-reading frames as well as 29 new ncRNAs were identified. Several novel riboswitches possibly functioning as upstream terminators were also found.

My thesis work compiles together a variety of novel RNA-mediated gene regulatory entities. A first coordinated transcriptional map of Listeria monocytogenes has been set up. My work has also revealed that the expression of the virulence regulator PrfA is controlled at several levels, indicating the importance of both the 5´-UTR and the coding RNA for regulated expression.

Place, publisher, year, edition, pages
Umeå, Sweden: Print & Media, 2010. 90 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1337
Keyword
Listeria monocytogenes, virulence, ncRNAs, PrfA, 5´-UTRs
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-33096 (URN)0346-6612 (ISBN)
Public defence
2010-05-12, Major Groove, Department of Molecular Biology, Umeå University, 10:00 (English)
Opponent
Supervisors
Available from: 2010-04-20 Created: 2010-04-12 Last updated: 2010-04-20Bibliographically approved

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Loh, EdmundJohansson, Jörgen

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Department of Molecular Biology (Faculty of Medicine)Molecular Infection Medicine Sweden (MIMS)Umeå Centre for Microbial Research (UCMR)
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