Journal
NUCLEIC ACIDS RESEARCH
Volume 40, Issue 4, Pages 1818-1827Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkr850
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Funding
- Magn. Bergvalls Stiftelse
- Umea University
- Swedish Research Council [K2007-57X-20355-01-3, K2011-56X-15144-08-6, 621-2009-5677]
- ERC [260764]
- The Danish Council for Independent Research / Natural Sciences [09-063992]
- European Research Council (ERC) [260764] Funding Source: European Research Council (ERC)
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Expression of virulence factors in the human bacterial 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), and is high at 37 degrees C and low at temperatures < 30 degrees C. In order to develop a thermoregulated translational expression system, the 5'-UTR and different lengths of the prfA-coding sequences were placed in front of lacZ. When expressed in Escherichia coli, the beta-galactosidase expression was directly correlated to the length of the prfA-coding mRNA lying in front of lacZ. A similar effect was detected with gfp as a reporter gene in both L. monocytogenes and E. coli, emphasizing the requirement of the prfA-coding RNA for maximal expression. In vitro transcription/translation and mutational analysis suggests a role for the first 20 codons of the native prfA-mRNA for maximal expression. By toe-print and RNA-probing analysis, a flexible hairpin-loop located immediately downstream of the start-codon was shown to be important for ribosomal binding. The present work determines the importance of an unstructured part of the 5'-coding region of the prfA-mRNA for efficient translation.
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