title: An unstructured 5'-coding region of the prfA mRNA is required for efficient translation.
abstract: 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.
authors: Department of Molecular Biology, Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, 90187 Umeå, Sweden.
authors: Edmund
authors: E
authors: Loh
authors: Faranak
authors: F
authors: Memarpour
authors: Karolis
authors: K
authors: Vaitkevicius
authors: Birgitte H
authors: BH
authors: Kallipolitis
authors: Jörgen
authors: J
authors: Johansson
authors: Berit
authors: B
authors: Sondén
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D003062:Codon; D017930:Genes, Reporter; D008089:Listeria monocytogenes / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009690:Nucleic Acid Conformation; D010454:Peptide Termination Factors / Q000096:biosynthesis / Q000235:genetics*; D014176:Protein Biosynthesis; D055550:Protein Stability; D020871:RNA Stability; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D010454:Peptide Termination Factors; C545356:PrfA protein, Listeria monocytogenes; D012333:RNA, Messenger