An unstructured 50-coding region of the prfA mRNA is required for efficient translation

Expression of virulence factors in the human bacter-ial pathogen Listeria monocytogenes is almost ex-clusively regulated by the transcriptional activator PrfA. The translation of prfA is controlled by a thermosensor located in the 50-untranslated RNA (UTR), and is high at 37C and low at temperatures <30C. In order to develop a thermoregulated trans-lational expression system, the 50-UTR and different lengths of the prfA-coding sequences were placed in front of lacZ. When expressed in Escherichia coli, the b-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 re-quirement of the prfA-coding RNA for maximal ex-pression. In vitro transcription/translation and mutational analysis suggests a role for the first 20 codons of the native prfA-mRNA for maximal ex-pression. By toe-print and RNA-probing analysis, a flexible hairpin-loop located immediately down-stream of the start-codon was shown to be import-ant for ribosomal binding. The present work determines the importance of an unstructured part of the 50-coding region of the prfA-mRNA for effi-cient translation