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

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.

In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa

“…Indeed, this assay could very well be adapted to any substrate because the RNA scaffold encapsulated in the OprM proteoliposomes can be modified. The RNA scaffold has a restriction site in which any nucleic acid material can be inserted 25 . Furthermore, DNA/RNA aptamers specific for an increasing number of targets are now available, and it has been shown that it is possible to rationally convert non-fluorescent aptamers into fluorescent reporters 26 .…”

Bioengineered non-coding RNA agent (BERA) in action

“…32,33 The tRNA-carried ncRNAs include a number of viral RNAs, RNA aptamers, hammerhead riboswitch RNAs, and human pre-miRNAs. [32][33][34][35][36][37][38][39] Nevertheless, levels of recombinant ncRNAs accumulated in cells are largely variable and inevitably dependent upon the structures and metabolic stabilities of chimeric RNAs. 34,35,40 An optimal ncRNA scaffold (OnRS) approach has been developed toward a more general, versatile, and robust high-yield and large-scale production of RNAi agents 40 (Table 1).…”

In vivo tmRNA protection by SmpB and pre-ribosome binding conformation in solution

“…With the idea that SmpB may protect aa-tmRNA from degradation, we decided to co-overproduce the A. aeolicus SmpB (aa-SmpB) and aa-tmRNA in LB medium. Both genes were cloned in our homemade plasmid pProRNA (Ponchon et al 2013). RNA transcription is constitutively controlled by the lpp promoter, while that of SmpB is controlled by the inducible lac promoter.…”

“…We recently reported the co-overproduction of the armored version of aa-tmRNA (Ar-aa-mRNA) with the MS2-His6 coat protein (138 aa) using the homemade p44K plasmid (Ponchon et al 2013). The Ar-aa-MLD was modified compared to wild type to incorporate, instead of the short stemloop of the aa-MLD, the 23-nt-long encapsidation signal stem-loop of the MS2 coat protein (Fig.…”

“…Cells transformed with pBSTNAV, pProRNA, or p44K plasmids were grown in either LB or 2XTY growth medium and harvested as described before (Ponchon et al 2013). The RNAs were recovered following protocols described by Ponchon et al (2009).…”