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

TmRNA is an abundant RNA in bacteria with tRNA and mRNA features. It is specialized in trans-translation, a translation rescuing system. We demonstrate that its partner protein SmpB binds the tRNA-like region (TLD) in vivo and chaperones the fold of the TLD-H2 region. We use an original approach combining the observation of tmRNA degradation pathways in a heterologous system, the analysis of the tmRNA digests by MS and NMR, and co-overproduction assays of tmRNA and SmpB. We study the conformation in solution of tmRNA alone or in complex with one SmpB before ribosome binding using SAXS. Our data show that Mg(2+) drives compaction of the RNA structure and that, in the absence of Mg(2+), SmpB has a similar effect albeit to a lesser extent. Our results show that tmRNA is intrinsically structured in solution with identical topology to that observed on complexes on ribosomes which should facilitate its subsequent recruitment by the 70S ribosome, free or preloaded with one SmpB molecule.

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.

Trapping conformational states along ligand-binding dynamics of peptide deformylase: the impact of induced fit on enzyme catalysis.

For several decades, molecular recognition has been considered one of the most fundamental processes in biochemistry. For enzymes, substrate binding is often coupled to conformational changes that alter the local environment of the active site to align the reactive groups for efficient catalysis and to reach the transition state. Adaptive substrate recognition is a well-known concept; however, it has been poorly characterized at a structural level because of its dynamic nature. Here, we provide a detailed mechanism for an induced-fit process at atomic resolution. We take advantage of a slow, tight binding inhibitor-enzyme system, actinonin-peptide deformylase. Crystal structures of the initial open state and final closed state were solved, as well as those of several intermediate mimics captured during the process. Ligand-induced reshaping of a hydrophobic pocket drives closure of the active site, which is finally "zipped up" by additional binding interactions. Together with biochemical analyses, these data allow a coherent reconstruction of the sequence of events leading from the encounter complex to the key-lock binding state of the enzyme. A "movie" that reconstructs this entire process can be further extrapolated to catalysis.

RNA-methyltransferase TrmA is a dual-specific enzyme responsible for C5-methylation of uridine in both tmRNA and tRNA.

In bacteria, trans-translation rescues stalled ribosomes by the combined action of tmRNA (transfer-mRNA) and its associated protein SmpB. The tmRNA 5' and 3' ends fold into a tRNA-like domain (TLD), which shares structural and functional similarities with tRNAs. As in tRNAs, the UUC sequence of the T-arm of the TLD is post-transcriptionally modified to m (5)UψC. In tRNAs of gram-negative bacteria, formation of m (5)U is catalyzed by the SAM-dependent methyltransferase TrmA, while formation of m (5)U at two different positions in rRNA is catalyzed by distinct site-specific methyltransferases RlmC and RlmD. Here, we show that m (5)U formation in tmRNAs is exclusively due to TrmA and should be considered as a dual-specific enzyme. The evidence comes from the lack of m (5)U in purified tmRNA or TLD variants recovered from an Escherichia coli mutant strain deleted of the trmA gene. Detection of m (5)U in RNA was performed by NMR analysis.

New peptide deformylase inhibitors and cooperative interaction: a combination to improve antibacterial activity.

OBJECTIVES: Bacterial drug resistance is a worrying public health problem and there is an urgent need for research and development to provide new antibacterial molecules. Peptide deformylase (PDF) is now a well-described intracellular target selected for the design of a new antibiotic group, PDF inhibitors (PDFIs). The initial bacterial susceptibility to an inhibitor of a cytoplasmic target is directly associated with the diffusion of the compound through the membrane barrier of Gram-negative bacteria and with its cytosolic accumulation at the required concentration. METHODS: We have recently demonstrated that the activity of different PDFIs is strongly dependent on the accumulation of the active molecules by using permeabilizing agents, efflux inhibitors or efflux-mutated strains. In this work we assessed various combination protocols using different putative inhibitors (PDFIs, methionine aminopeptidase inhibitors etc.) to improve antibacterial activity against various resistant Gram-negative bacteria. RESULTS: The maximum effect was observed when combining actinonin with a dual inhibitor of methionine aminopeptidase and PDF, this molecule being also able to interact with the target while actinonin is bound to the PDF active site. CONCLUSIONS: Such a combination of inhibitors acting on two tightly associated metabolic steps results in a cooperative effect on bacterial cells and opens an original way to combat multidrug-resistant bacteria.

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.

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 DeltasmpB 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.

Coexpression and Copurification of RNA-Protein Complexes in Escherichia coli.

For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. We previously devised a generic approach that allows for efficient in vivo expression of recombinant RNA in Escherichia coli. We have extended the "tRNA scaffold" method to RNA-protein coexpression in order to express and purify RNA by affinity in native condition. As a proof of concept, we present the expression and the purification of the AtRNA-mala in complex with the MS2 coat protein.

A unique conformation of the anticodon stem-loop is associated with the capacity of tRNAfMet to initiate protein synthesis.

In all organisms, translational initiation takes place on the small ribosomal subunit and two classes of methionine tRNA are present. The initiator is used exclusively for initiation of protein synthesis while the elongator is used for inserting methionine internally in the nascent polypeptide chain. The crystal structure of Escherichia coli initiator tRNA(f)(Met) has been solved at 3.1 A resolution. The anticodon region is well-defined and reveals a unique structure, which has not been described in any other tRNA. It encompasses a Cm32*A38 base pair with a peculiar geometry extending the anticodon helix, a base triple between A37 and the G29-C41 pair in the major groove of the anticodon stem and a modified stacking organization of the anticodon loop. This conformation is associated with the three GC basepairs in the anticodon stem, characteristic of initiator tRNAs and suggests a mechanism by which the translation initiation machinery could discriminate the initiator tRNA from all other tRNAs.

Design of tRNA(Lys)3 ligands: fragment evolution and linker selection guided by NMR spectroscopy.

A fragment-based approach for the synthesis of ligands of tRNA(Lys) (3), the HIV reverse-transcription primer, is described. The use of NMR spectroscopy has proved to be very useful in this approach, not only to detect low-affinity complexes between small compounds and RNA, but also to provide information on their binding mode and on the way they can be connected. This NMR-spectroscopy-guided analysis enabled us to design micromolar ligands after the optimisation and connection of millimolar fragments with an appropriate linker. The influence of the linker region on the binding affinity and selectivity outlines the importance of having a flexible assemblage strategy with a variety of linkers in such an approach.

NMR structure of the Aquifex aeolicus tmRNA pseudoknot PK1: new insights into the recoding event of the ribosomal trans-translation.

The transfer-messenger RNA (tmRNA) pseudoknot PK1 is essential for bacterial trans-translation, a ribosomal rescue mechanism. We report the solution structure of PK1 from Aquifex aeolicus, which despite an unprecedented small number of nucleotides and thus an unprecented compact size, displays a very high thermal stability. Several unusual structural features account for these properties and indicate that PK1 belongs to the class of ribosomal frameshift pseudoknots. This suggests a similarity between the mechanism of programmed ribosomal frameshifting and trans-translation.

New insights into the formation of HIV-1 reverse transcription initiation complex.

HIV-1 reverse transcriptase uses the host tRNA3(Lys) as a primer for the synthesis of the minus DNA strand. The first event in viral replication is thus the annealing of tRNA to the primer binding site (PBS) in the 5' UTR of the viral RNA. This event requires a major RNA rearrangement which is chaperoned by the viral NC protein. The binding of NC to nucleic acids is essentially non-specific, however, NC is known to bind selectively to hairpins located in the 5' region of the viral RNA. In a previous study, using an NMR approach in which the reaction is slowed down by controlling temperature, we were able to follow details in this RNA unfolding/refolding process and to uncover an intermediate state. We showed that annealing initiates at the junction between the acceptor and the TPsiC stems, and that, at physiological temperature, complete annealing is reached only in the presence of NC, probably when the zinc fingers contact the TPsiC/D loops. In the present work, we have refined our model of the formation of the tRNA3(Lys)/PBS duplex. First, we show that annealing can initiate both from the single-stranded CCA 3'-end bases of the acceptor stem and from the bases in the TPsiC stem. Secondly, by NMR and fluorescence spectroscopy, we have studied the complex between the NC protein and RNA hairpins that mimic the D and T arms of the tRNA3(Lys). Interestingly, the NC protein shows strong and specific binding to the D arm of tRNA3(Lys), which could explain the overall annealing mechanism.

Discovery and refinement of a new structural class of potent peptide deformylase inhibitors.

New classes of antibiotics are urgently needed to counter increasing levels of pathogen resistance. Peptide deformylase (PDF) was originally selected as a specific bacterial target, but a human homologue, the inhibition of which causes cell death, was recently discovered. We developed a dual-screening strategy for selecting highly effective compounds with low inhibition effect against human PDF. We selected a new scaffold in vitro that discriminated between human and bacterial PDFs. Analyses of structure-activity relationships identified potent antibiotics such as 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide (6b) with the same mode of action in vivo as previously identified PDF inhibitors but without the apoptotic effects of these inhibitors in human cells.

Solution NMR structure of the SH3 domain of human nephrocystin and analysis of a mutation-causing juvenile nephronophthisis.

Human nephrocystin is a protein associated with juvenile NPH, an autosomal recessive, inherited kidney disease responsible for chronic renal failure in children. It contains an SH3 domain involved in signaling pathways controlling cell adhesion and cytoskeleton organization. The solution structure of this domain was solved by triple resonance NMR spectroscopy. Within the core, the structure is similar to those previously reported for other SH3 domains but exhibits a number of specific noncanonical features within the polyproline ligand binding site. Some of the key conserved residues are missing, and the N-Src loop exhibits an unusual twisted geometry, which results in a narrowing of the binding groove. This is induced by the replacement of a conserved Asp, Asn, or Glu residue by a Pro at one side of the N-Src loop. A systematic survey of other SH3 domains also containing a Pro at this position reveals that most of them belong to proteins involved in cell adhesion or motility. A variant of this domain, which carries a point mutation causing NPH, was also analyzed. This change, L180P, although it corresponds to a nonconserved and solvent-exposed position, causes a complete loss of the tertiary structure. Similar effects are also observed with the L180A variant. This could be a context-dependent effect resulting from an interaction between neighboring charged side-chains.

NMR-based identification of peptides that specifically recognize the d-arm of tRNA.

Human tRNA3(Lys) is used by HIV virus as a primer for the reverse transcription of its genome. The 18 nucleotides at the 3'-end of the tRNA3(Lys) are hybridized to a complementary sequence of the viral RNA called the primer-binding site. A screen against the human tRNA3(Lys) over a peptide library designed to target RNA has been performed. Of the 175 hexapeptides tested, three were found to bind to the d-stem of tRNA3(Lys). Alanine-scanning was used to define the determinants of the interaction between the peptides and tRNA3(Lys). They also bind to two other tested tRNAs, also at the level of the d-stem and loop, although the nucleotide sequence of the stem differs in one of them. These short peptides thus recognize specific structural features within the d-stem and loop of tRNAs. Associated with other pharmacophores, they could be useful to design optimized ligands targeting specific tRNAs such as retroviral replication primers.

Self-organisation of an oligodeoxynucleotide containing the G- and C-rich stretches of the direct repeats of the human mitochondrial DNA.

Stretches of cytosines and guanosines have been shown in vitro to adopt non-canonical structures known as i-motifs and G-quartets, respectively. When combined, such sequences are expected to either retain their structure or form duplexes or triple helices. All these structures may occur in vivo whenever the sequence criteria are met. Such stretches are present in the circular genome of human mitochondria, as two 10 nucleotide-long perfect tandem direct repeats (DR1 and DR2). The DR1 and DR2 repeats are G-rich on the heavy strand and C-rich on the light strand. Previous results suggested that during replication, transient formation of a parallel GGC triple helix between the neo-synthesised G-rich DR1 and the double-stranded homologous DR2 could be involved in a rearrangement process leading to genome instability. In order to get structural insights into the interaction between the two repeats, we have studied by nuclear magnetic resonance (NMR) the assembly properties of a 24-mer oligodeoxyribonucleotide in which the C- and G-rich segments of the DRs are covalently tethered by a TTTT linker. We show here that this 24-mer self-associates into a triplex-containing symmetrical tetramer. The core of the structure is composed of anti-parallel Watson-Crick (WC) base pairs. Two additional strands are hydrogen-bonded to the Hoogsteen side of the Gs, thus forming CGC(+) triple helices, with G-rich ends folding into G-quartets. These results suggest that such structures could occur when the two DRs are put to close proximity in a biological context.

The tRNA-like domains of E coli and A.aeolicus transfer-messenger RNA: structural and functional studies.

Transfer-messenger RNA (tmRNA, 10Sa RNA or ssrA) acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis. The understanding at molecular level of this ubiquitous quality control system in eubacteria requires structural information. Here, we describe the purification and structural analysis of a functional fragment of both Aquifex aeolicus and Escherichia coli tmRNA, recapitulating their tRNA-like domain, which were expressed in vivo from synthetic genes. Both recombinant RNA are correctly processed at both 5' and 3' ends and are produced in quantities suitable for structural analysis by NMR and/or X-ray crystallography. The sequence and solution structure of the tRNA-like domains were analysed by various methods including structural mapping with chemical and enzymatic probes and 2D NMR spectroscopy. The minimalist RNAs contain two post-transcriptional base modifications, 5-methyluridine and pseudouridine, as the full-length tmRNA. Both RNAs fold into three stems, a D-analogue, a T-loop and a GAAA tetra-loop. 2D NMR analysis of the imino proton resonances of both RNAs allowed the assignment of the three stems and of a number of tertiary interactions. It shows the existence of interactions between the TPsiC-loop and the D-analogue, exhibiting a number of similarities and also differences with the canonical tRNA fold, indicating that RNA tertiary interactions can be modulated according to the sequence and secondary structure contexts. Furthermore, the E.coli minimalist RNA is aminoacylatable with alanine with a catalytic efficiency an order of magnitude higher than that for full-length tmRNA.

NMR structure of bacterial ribosomal protein l20: implications for ribosome assembly and translational control.

L20 is a specific protein of the bacterial ribosome, which is involved in the early assembly steps of the 50S subunit and in the feedback control of the expression of its own gene. This dual function involves specific interactions with either the 23S rRNA or its messenger RNA. The solution structure of the free Aquifex aeolicus L20 has been solved. It is composed of an unstructured N-terminal domain comprising residues 1-58 and a C-terminal alpha-helical domain. This is in contrast with what is observed in the bacterial 50S subunit, where the N-terminal region folds as an elongated alpha-helical region. The solution structure of the C-terminal domain shows that several solvent-accessible, conserved residues are clustered on the surface of the molecule and are probably involved in RNA recognition. In vivo studies show that this domain is sufficient to repress the expression of the cistrons encoding L35 and L20 in the IF3 operon. The ability of L20 C-terminal domain to specifically recognise RNA suggests an assembly mechanism for L20 into the ribosome. The pre-folded C-terminal domain would make a primary interaction with a specific site on the 23S rRNA. The N-terminal domain would then fold within the ribosome, participating in its correct 3D assembly.

Expression and Purification of RNA-Protein Complexes in Escherichia coli.

For structural, biochemical or pharmacological studies, it is required to have pure RNA in large quantities. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. We have extended the "tRNA scaffold" method to RNA/protein co-expression in order to express and purify RNA by affinity in native condition. As a proof-of-concept, we present the expression and the purification of the AtRNA-mala in complex with the MS2 coat protein.