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Co-expression of RNA-protein complexes in Escherichia coli and applications to RNA biology.

Ponchon L, Catala M, Seijo B, El Khouri M, Dardel F, Nonin-Lecomte S, Tisné C - Nucleic Acids Res. (2013)

Bottom Line: RNA has emerged as a major player in many cellular processes.We show that this last application easily provides pure material for crystallographic studies.The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

View Article: PubMed Central - PubMed

Affiliation: CNRS, UMR 8015, Laboratoire de Cristallographie et RMN biologiques, 4 avenue de l'Observatoire, 75006 Paris, France and Université Paris Descartes, Sorbonne Paris Cité, UMR 8015, Laboratoire de Cristallographie et RMN biologiques, 4 avenue de l'Observatoire, 75006 Paris, France.

ABSTRACT
RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA-protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA-His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

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The A. aeolicus AtmRNA encapsulated in MS2 coat protein pseudo-particles is protected against nucleases. (A) Secondary scheme of A. aeolicus AtmRNA showing the MS2 translational operator, the tRNA-like domain and the MLR in grey. (B) Overexpression of the AtmRNA alone in E. coli JM101 (left part of the upper gel) and co-expressed with the MS2 coat protein (right part of the upper gel): crude RNA minipreps were analysed by electrophoresis on a 10% polyacrylamide-urea gel and RNA visualized with SYBR Safe (upper part). Proteins in the supernatant after bacteria lysis were visualized by SDS–PAGE and Coomassie Brilliant Blue staining (lower gel) before and after IPTG induction (−/+). The black triangle indicates the AtmRNA band and the grey triangle the MS2 coat protein band. The AtmRNA/MS2 coat protein pair was cloned into pBSTNAV and pACYT2 plasmids, respectively.
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gkt576-F4: The A. aeolicus AtmRNA encapsulated in MS2 coat protein pseudo-particles is protected against nucleases. (A) Secondary scheme of A. aeolicus AtmRNA showing the MS2 translational operator, the tRNA-like domain and the MLR in grey. (B) Overexpression of the AtmRNA alone in E. coli JM101 (left part of the upper gel) and co-expressed with the MS2 coat protein (right part of the upper gel): crude RNA minipreps were analysed by electrophoresis on a 10% polyacrylamide-urea gel and RNA visualized with SYBR Safe (upper part). Proteins in the supernatant after bacteria lysis were visualized by SDS–PAGE and Coomassie Brilliant Blue staining (lower gel) before and after IPTG induction (−/+). The black triangle indicates the AtmRNA band and the grey triangle the MS2 coat protein band. The AtmRNA/MS2 coat protein pair was cloned into pBSTNAV and pACYT2 plasmids, respectively.

Mentions: In our ongoing research on trans-translation, we were trying to produce the A. aeolicus tmRNA using the pBSTNAV vector in E. coli. Trans-translation is a highly sophisticated process in bacteria that recycles ribosomes stalled on defective mRNAs and adds a short tag-peptide to the C-terminus of the incomplete polypeptide as degradation signal [for review, see (48)]. The process of trans-translation uses the tmRNA, which is a unique molecule with dual tRNA and mRNA functions (Figure 4A). The A. aeolicus tmRNA (A. aeolicus tmRNA) is a multidomain RNA of 347 nt. It comprises two functional domains, the tRNA domain partially mimicking a tRNA and the mRNA-like region (MLR), which encodes the tag peptide, surrounded by four pseudo-knots. This molecule is, therefore, a case study for our ‘tRNA scaffold’ approach, as it naturally contains a tRNA domain. Although the overproduction is good, the A. aeolicus tmRNA undergoes systematic degradation by nucleases during its expression in E. coli (Figure 4B). Two bands of approximately the same intensities are observed on a polyacrylamide-urea gel. The lower band evidences a truncated form resulting from the cleavage by nucleases in the cytoplasm of E. coli during its overproduction.Figure 4.


Co-expression of RNA-protein complexes in Escherichia coli and applications to RNA biology.

Ponchon L, Catala M, Seijo B, El Khouri M, Dardel F, Nonin-Lecomte S, Tisné C - Nucleic Acids Res. (2013)

The A. aeolicus AtmRNA encapsulated in MS2 coat protein pseudo-particles is protected against nucleases. (A) Secondary scheme of A. aeolicus AtmRNA showing the MS2 translational operator, the tRNA-like domain and the MLR in grey. (B) Overexpression of the AtmRNA alone in E. coli JM101 (left part of the upper gel) and co-expressed with the MS2 coat protein (right part of the upper gel): crude RNA minipreps were analysed by electrophoresis on a 10% polyacrylamide-urea gel and RNA visualized with SYBR Safe (upper part). Proteins in the supernatant after bacteria lysis were visualized by SDS–PAGE and Coomassie Brilliant Blue staining (lower gel) before and after IPTG induction (−/+). The black triangle indicates the AtmRNA band and the grey triangle the MS2 coat protein band. The AtmRNA/MS2 coat protein pair was cloned into pBSTNAV and pACYT2 plasmids, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753655&req=5

gkt576-F4: The A. aeolicus AtmRNA encapsulated in MS2 coat protein pseudo-particles is protected against nucleases. (A) Secondary scheme of A. aeolicus AtmRNA showing the MS2 translational operator, the tRNA-like domain and the MLR in grey. (B) Overexpression of the AtmRNA alone in E. coli JM101 (left part of the upper gel) and co-expressed with the MS2 coat protein (right part of the upper gel): crude RNA minipreps were analysed by electrophoresis on a 10% polyacrylamide-urea gel and RNA visualized with SYBR Safe (upper part). Proteins in the supernatant after bacteria lysis were visualized by SDS–PAGE and Coomassie Brilliant Blue staining (lower gel) before and after IPTG induction (−/+). The black triangle indicates the AtmRNA band and the grey triangle the MS2 coat protein band. The AtmRNA/MS2 coat protein pair was cloned into pBSTNAV and pACYT2 plasmids, respectively.
Mentions: In our ongoing research on trans-translation, we were trying to produce the A. aeolicus tmRNA using the pBSTNAV vector in E. coli. Trans-translation is a highly sophisticated process in bacteria that recycles ribosomes stalled on defective mRNAs and adds a short tag-peptide to the C-terminus of the incomplete polypeptide as degradation signal [for review, see (48)]. The process of trans-translation uses the tmRNA, which is a unique molecule with dual tRNA and mRNA functions (Figure 4A). The A. aeolicus tmRNA (A. aeolicus tmRNA) is a multidomain RNA of 347 nt. It comprises two functional domains, the tRNA domain partially mimicking a tRNA and the mRNA-like region (MLR), which encodes the tag peptide, surrounded by four pseudo-knots. This molecule is, therefore, a case study for our ‘tRNA scaffold’ approach, as it naturally contains a tRNA domain. Although the overproduction is good, the A. aeolicus tmRNA undergoes systematic degradation by nucleases during its expression in E. coli (Figure 4B). Two bands of approximately the same intensities are observed on a polyacrylamide-urea gel. The lower band evidences a truncated form resulting from the cleavage by nucleases in the cytoplasm of E. coli during its overproduction.Figure 4.

Bottom Line: RNA has emerged as a major player in many cellular processes.We show that this last application easily provides pure material for crystallographic studies.The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

View Article: PubMed Central - PubMed

Affiliation: CNRS, UMR 8015, Laboratoire de Cristallographie et RMN biologiques, 4 avenue de l'Observatoire, 75006 Paris, France and Université Paris Descartes, Sorbonne Paris Cité, UMR 8015, Laboratoire de Cristallographie et RMN biologiques, 4 avenue de l'Observatoire, 75006 Paris, France.

ABSTRACT
RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA-protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA-His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

Show MeSH
Related in: MedlinePlus