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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°C and low at temperatures <30°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 β-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.

Original publication

DOI

10.1093/nar/gkr850

Type

Journal article

Journal

Nucleic Acids Res

Publication Date

02/2012

Volume

40

Pages

1818 - 1827

Keywords

Bacterial Proteins, Codon, Genes, Reporter, Listeria monocytogenes, Mutation, Nucleic Acid Conformation, Peptide Termination Factors, Protein Biosynthesis, Protein Stability, RNA Stability, RNA, Messenger, Ribosomes