An unstructured 5′-coding region of the prfA mRNA is required for efficient translation

Department of Molecular Biology, Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, 90187 Umeå, Sweden.
Nucleic Acids Research (Impact Factor: 9.11). 11/2011; 40(4):1818-27. DOI: 10.1093/nar/gkr850
Source: PubMed


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.

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    • "Besides the promoters, the 5′-untranslated regions (5′-UTRs) of bacterial mRNA are also known to play important regulatory roles in gene expression, which possibly occur at the transcriptional, post-transcriptional, or translational levels [9]. Extremely diverse mechanisms are employed by the cis-acting RNA regulatory elements in 5′-UTRs to strictly adjust the cellular levels of their downstream genes, including: (i) the ability of many 5′-UTRs to recognize a specific regulatory signal, such as T-boxes, riboswitches and RNA thermometers [10]–[12]; (ii) the capability of some 5′-UTRs to provide binding sites for small regulatory RNAs [9], [13]; and (iii) more 5′-UTRs being able to regulate the expression of the downstream gene, presumably by RNase III-mediated cleavage modification [14], preventing degradation of the mRNA [15], or other unknown mechanisms. Therefore, besides promoters, some 5′-UTR DNA regions have a significant applied potential in molecular biology research and improvement of recombinant protein expression [9], [12], [16], [17]. "
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    PLoS ONE 05/2013; 8(5):e62960. DOI:10.1371/journal.pone.0062960 · 3.23 Impact Factor
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    • "In this report we describe a systematic comparison of both positively and negatively regulated expression systems. Being aware of the influence of the 5′ end of the coding region on expression [29,30], we intentionally chose to use model genes with native 5′ ends as opposed to commonly used regions encoding N-terminal detection tags or solubility-enhancing fusion partners. The expression analyses were carried out at both the transcript and the protein level (activity assays and total protein), and we also included a flow cytometry based analysis of expression in individual cells. "
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    • "The authors of this study found that if the lcrF thermosensor is mutated to create a permanently open or permanently closed conformation, the virulence of Y. pseudotuberculosis is reduced in a mouse model of Yersiniosis, which suggests that the ability to fine tune the translation of LcrF – either up or down – is critical to the virulence of this pathogen. So far only one other virulence-associated thermosensor has been identified in a bacterial pathogen: the prfA gene (a positive regulator of listeriolysin) of Listeria monocytogenes (Johansson et al., 2002; Loh et al., 2012). Given the major contribution of temperature to the biology of Yersinia, it is possible that there may be other thermosensors that remain unidentified, including those that regulate virulence genes. "
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    Frontiers in Cellular and Infection Microbiology 11/2012; 2:129. DOI:10.3389/fcimb.2012.00129 · 3.72 Impact Factor
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