Luc Ponchon

A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold

RNA production using in vivo transcription by Escherichia coli allows preparation of milligram quantities of RNA for biochemical, biophysical and structural investigations. We describe here a generic protocol for the overproduction and purification of recombinant RNA using liquid chromatography. The strategy utilizes a transfer RNA (tRNA) as a scaffold that can be removed from the RNA of interest by digestion of the fusion RNA at a designed site by RNase H. The tRNA scaffold serves to enhance the stability and to promote the proper expression of its fusion partners. This protocol describes how to construct a tRNA fusion RNA expression vector; to conduct a pilot experiment to assess the yield of the recombinant RNA both before and after processing of the fusion RNA by RNase H; and to purify the target RNA on a large scale for structural or functional studies. This protocol greatly facilitates production of RNA in a time frame of ∼3 weeks from design to purification. As compared with in vitro methods (transcription, chemical synthesis), this approach is simple, cheap and well suited for large-scale expression and isotope labeling.

Ribosome hijacking: a role for small protein B during trans‐translation

Tight recognition of codon–anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. In eubacteria, translational surveillance and ribosome rescue are performed by the ‘tmRNA–SmpB’ system (transfer messenger RNA–small protein B). Remarkably, entry and accommodation of aminoacylated‐tmRNA into stalled ribosomes occur without a codon–anticodon interaction but in the presence of SmpB. Here, we show that within a stalled ribosome, SmpB interacts with the three universally conserved bases G530, A1492 and A1493 that form the 30S subunit decoding centre, in which canonical codon–anticodon pairing occurs. The footprints at positions A1492 and A1493 of a small decoding centre, as well as on a set of conserved SmpB amino acids, were identified by nuclear magnetic resonance. Mutants at these residues display the same growth defects as for ΔsmpB strains. The SmpB protein has functional and structural similarities with initiation factor 1, and is proposed to be a functional mimic of the pairing between a codon and an anticodon.

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

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.

Large scale expression and purification of recombinant RNA in Escherichia coli

Stable, folded RNA are involved in many key cellular processes and can be used as tools for biological, pharmacological and/or molecular design studies. However, their widespread use has been somewhat limited by their fragile nature and by the difficulties associated with their production on a large scale, which were limited to in vitro methods. This work reviews the novel techniques recently developed that allow efficient expression of recombinant RNA in vivo in Escherichia coli. Based on the extensive data available on the genetic and metabolic mechanisms of this model organism, conditions for optimal production can be derived. Combined with a large repertoire of RNA motifs which can be assembled by recombinant DNA techniques, this opens the way to the modular design of RNA molecules with novel properties.

The Endonuclease Domain of Bacteriophage Terminases Belongs to the Resolvase/Integrase/Ribonuclease H Superfamily A BIOINFORMATICS ANALYSIS VALIDATED BY A FUNCTIONAL STUDY ON BACTERIOPHAGE T5

Bacteriophage terminases are essential molecular motors involved in the encapsidation of viral DNA. They are hetero-multimers whose large subunit encodes both ATPase and endonuclease activities. Although the ATPase domain is well characterized from sequence and functional analysis, the C-terminal region remains poorly defined. We describe sequence-structure comparisons of the endonuclease region of various bacteriophages that revealed new sequence similarities shared by this region and the Holliday junction resolvase RuvC and to a lesser extent the HIV integrase and the ribonuclease H. Extensive sequence comparison and motif refinement led to a common signature of terminases and resolvases with three conserved acidic residues engaged in catalytic activity. Sequence analyses were validated by in vivo and in vitro functional assays showing that the nuclease activity of the endonuclease domain of bacteriophage T5 terminase was abolished by mutation of any of the three predicted catalytic aspartates. Overall, these data suggest that the endonuclease domains of terminases operate autonomously and that they adopt a fold similar to that of resolvases and share the same divalent cation-dependent enzymatic mechanism.

The m1A58 modification in eubacterial tRNA: An overview of tRNA recognition and mechanism of catalysis by TrmI

The enzymes of the TrmI family catalyze the formation of the m(1)A58 modification in tRNA. We previously solved the crystal structure of the Thermus thermophilus enzyme and conducted a biophysical study to characterize the interaction between TrmI and tRNA. TrmI enzymes are active as a tetramer and up to two tRNAs can bind to TrmI simultaneously. In this paper, we present the structures of two TrmI mutants (D170A and Y78A). These residues are conserved in the active site of TrmIs and their mutations result in a dramatic alteration of TrmI activity. Both structures of TrmI mutants revealed the flexibility of the N-terminal domain that is probably important to bind tRNA. The structure of TrmI Y78A catalytic domain is unmodified regarding the binding of the SAM co-factor and the conformation of residues potentially interacting with the substrate adenine. This structure reinforces the previously proposed role of Y78, i.e. stabilize the conformation of the A58 ribose needed to hold the adenosine in the active site. The structure of the D170A mutant shows a flexible active site with one loop occupying in part the place of the co-factor and the second loop moving at the entrance to the active site. This structure and recent data confirms the central role of D170 residue binding the amino moiety of SAM and the exocyclic amino group of adenine. Possible mechanisms for methyl transfer are then discussed.

Selective RNase H Cleavage of Target RNAs from a tRNA Scaffold

In vivo overproduction of tRNA chimeras yields an RNA insert within a tRNA scaffold. For some applications, it may be necessary to discard the scaffold. Here we present a protocol for selective cleavage of the RNA of interest from the tRNA scaffold, using RNase H and two DNA oligonucleotides. After cleavage, we show that the RNA of interest can be isolated in a one-step purification. This method has, in particular, applications in structural investigations of RNA.

Backbone resonance assignment of the human uracil DNA glycosylase-2

The HIV-1 viral protein R (Vpr) is incorporated into virus particle during budding suggesting that its presence in the mature virion is required in the early steps of the virus life cycle in newly infected cells. Vpr is released into the host cell cytoplasm to participate to the translocation of the preintegration complex (PIC) into the nucleus for integration of the viral DNA into the host genome. Actually, Vpr plays a key role in the activation of the transcription of the HIV-1 long terminal repeat (LTR), mediates cell cycle arrest in G2 to M transition, facilitates apoptosis and controls the fidelity of reverse transcription. Moreover, Vpr drives the repair enzyme uracil DNA glycosylase (UNG2) towards degradation. UNG2 has a major role in “Base excision repair” (BER) whose main function is to maintain genome integrity by controlling DNA uracilation. The interaction of Vpr with the cellular protein UNG2 is a key event in various stages of retroviral replication and its role remains to be defined. We have performed the structural study of UNG2 by NMR and we report its (1HN, 15N, 13Cα, 13Cβ and 13C′) chemical shift backbone assignment and its secondary structure in solution as predicted by TALOS-N. We aim to determine with accuracy by NMR, the residues of UNG2 interacting with Vpr, characterize their interaction and use the local structure of UNG2 and its interface with Vpr to propose potential ligands disturbing this interaction.

Purification of RNA Expressed In Vivo Inserted in a tRNA Scaffold

For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. In vitro transcription or chemical synthesis are the principal methods to produce RNA. Here, we describe an alternative method allowing RNA production in bacteria and its purification by liquid chromatography. In a few days, between 10 and 100 mg of pure RNA are obtained with this technique.