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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Available from: Birgitte H Kallipolitis
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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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    ABSTRACT: In bacteria, both promoters and 5'-untranslated regions (5'-UTRs) of mRNAs play vital regulatory roles in gene expression. In this study, we identified 1203 active promoter candidates in Bacillus thuringiensis through analysis of the genome-wide TSSs based on the transcriptome data. There were 11 types of σ-factor and 34 types of transcription factor binding sites found in 723 and 1097 active promoter candidates, respectively. Moreover, within the 1203 transcriptional units (TUs), most (52%) of the 5'-UTRs were 10-50 nucleotides in length, 12.8% of the TUs had a long 5'-UTR greater than 100 nucleotides in length, and 16.3% of the TUs were leaderless. We then selected 20 active promoter candidates combined with the corresponding 5'-UTR DNA regions to screen the highly active promoter-5'-UTR DNA region complexes with different characteristics. Our results demonstrate that among the 20 selected complexes, six were able to exert their functions throughout the life cycle, six were specifically induced during the early-stationary phase, and four were specifically activated during the mid-stationary phase. We found a direct corresponding relationship between σ-factor-recognized consensus sequences and complex activity features: the great majority of complexes acting throughout the life cycle possess σ(A)-like consensus sequences; the maximum activities of the σ(F)-, σ(E)-, σ(G)-, and σ(K)-dependent complexes appeared at 10, 14, 16, and 22 h under our experimental conditions, respectively. In particular, complex Phj3 exhibited the strongest activity. Several lines of evidence showed that complex Phj3 possessed three independent promoter regions located at -251∼-98, -113∼-31, and -54∼+14, and that the 5'-UTR +1∼+118 DNA region might be particularly beneficial to both the stability and translation of its downstream mRNA. Moreover, Phj3 successfully overexpressed the active β-galactosidase and turbo-RFP, indicating that Phj3 could be a proper regulatory element for overexpression of proteins in B. thuringiensis. Therefore, our efforts contribute to molecular biology research and the biotechnological application of B. thuringiensis.
    Full-text · Article · May 2013 · PLoS ONE
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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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    ABSTRACT: Background Production of recombinant proteins in bacteria for academic and commercial purposes is a well established field; however the outcomes of process developments for specific proteins are still often unpredictable. One reason is the limited understanding of the performance of expression cassettes relative to each other due to different genetic contexts. Here we report the results of a systematic study aiming at exclusively comparing commonly used regulator/promoter systems by standardizing the designs of the replicon backbones. Results The vectors used in this study are based on either the RK2- or the pMB1- origin of replication and contain the regulator/promoter regions of XylS/Pm (wild-type), XylS/Pm ML1-17 (a Pm variant), LacI/PT7lac, LacI/Ptrc and AraC/PBAD to control expression of different proteins with various origins. Generally and not unexpected high expression levels correlate with high replicon copy number and the LacI/PT7lac system generates more transcript than all the four other cassettes. However, this transcriptional feature does not always lead to a correspondingly more efficient protein production, particularly if protein functionality is considered. In most cases the XylS/Pm ML1-17 and LacI/PT7lac systems gave rise to the highest amounts of functional protein production, and the XylS/Pm ML1-17 is the most flexible in the sense that it does not require any specific features of the host. The AraC/PBAD system is very good with respect to tightness, and a commonly used bioinformatics prediction tool (RBS calculator) suggested that it has the most translation-efficient UTR. Expression was also studied by flow cytometry in individual cells, and the results indicate that cell to cell heterogeneity is very relevant for understanding protein production at the population level. Conclusions The choice of expression system needs to be evaluated for each specific case, but we believe that the standardized vectors developed for this study can be used to more easily identify the nature of case-specific bottlenecks. By then taking into account the relevant characteristics of each expression cassette it will be easier to make the best choice with respect to the goal of achieving high levels of protein expression in functional or non-functional form.
    Full-text · Article · Mar 2013 · Microbial Cell Factories
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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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    ABSTRACT: Proper regulation of gene expression is required by bacterial pathogens to respond to continually changing environmental conditions and the host response during the infectious process. While transcriptional regulation is perhaps the most well understood form of controlling gene expression, recent studies have demonstrated the importance of post-transcriptional mechanisms of gene regulation that allow for more refined management of the bacterial response to host conditions. Yersinia species of bacteria are known to use various forms of post-transcriptional regulation for control of many virulence-associated genes. These include regulation by cis- and trans-acting small non-coding RNAs, RNA-binding proteins, RNases, and thermoswitches. The effects of these and other regulatory mechanisms on Yersinia physiology can be profound and have been shown to influence type III secretion, motility, biofilm formation, host cell invasion, intracellular survival and replication, and more. In this review, we discuss these and other post-transcriptional mechanisms and their influence on virulence gene regulation, with a particular emphasis on how these processes influence the virulence of Yersinia in the host.
    Full-text · Article · Nov 2012 · Frontiers in Cellular and Infection Microbiology
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