Solution X-ray scattering highlights discrepancies in Plasmodium multi-aminoacyl-tRNA synthetase complexes.

tRip is a tRNA import protein specific to Plasmodium, the causative agent of malaria. In addition to its membrane localization and tRNA trafficking properties, tRip has the capacity to associate with three aminoacyl-tRNA synthetases (aaRS), the glutamyl- (ERS), glutaminyl- (QRS), and methionyl- (MRS) tRNA synthetases. In eukaryotes, such multi-aaRSs complexes (MSC) regulate the moonlighting activities of aaRSs. In Plasmodium, tRip and the three aaRSs all contain an N-terminal GST-like domain involved in the assembly of two independent complexes: the Q-complex (tRip:ERS:QRS) and the M-complex (tRip:ERS:MRS) with a 2:2:2 stoichiometry and in which the association of the GST-like domains of tRip and ERS (tRip-N:ERS-N) is central. In this study, the crystal structure of the N-terminal GST-like domain of ERS was solved and made possible further investigation of the solution architecture of the Q- and M-complexes by small-angle x-ray scattering (SAXS). This strategy relied on the engineering of a tRip-N-ERS-N chimeric protein to study the structural scaffold of both Plasmodium MSCs and confirm the unique homodimerization pattern of tRip in solution. The biological impact of these structural arrangements is discussed.

Identification of host tRNAs preferentially recognized by the Plasmodium surface protein tRip.

Malaria is a life-threatening and devastating parasitic disease. Our previous work showed that parasite development requires the import of exogenous transfer RNAs (tRNAs), which represents a novel and unique form of host-pathogen interaction, as well as a potentially druggable target. This import is mediated by tRip (tRNA import protein), a membrane protein located on the parasite surface. tRip displays an extracellular domain homologous to the well-characterized OB-fold tRNA-binding domain, a structural motif known to indiscriminately interact with tRNAs. We used MIST (Microarray Identification of Shifted tRNAs), a previously established in vitro approach, to systematically assess the specificity of complexes between native Homo sapiens tRNAs and recombinant Plasmodium falciparum tRip. We demonstrate that tRip unexpectedly binds to host tRNAs with a wide range of affinities, suggesting that only a small subset of human tRNAs is preferentially imported into the parasite. In particular, we show with in vitro transcribed constructs that tRip does not bind specific tRNAs solely based on their primary sequence, hinting that post-transcriptional modifications modulate the formation of our host/parasite molecular complex. Finally, we discuss the potential utilization of the most efficient tRip ligands for the translation of the parasite's genetic information.

Structural Analysis of RNA by Small-Angle X-ray Scattering.

Over the past two decades small-angle X-ray scattering (SAXS) has become a popular method to characterize solutions of biomolecules including ribonucleic acid (RNA). In an integrative structural approach, SAXS is complementary to crystallography, NMR, and electron microscopy and provides information about RNA architecture and dynamics. This chapter highlights the practical advantages of combining size-exclusion chromatography and SAXS at synchrotron facilities. It is illustrated by practical case studies of samples ranging from single hairpins and tRNA to a large IRES. The emphasis is also put on sample preparation which is a critical step of SAXS analysis and on optimized protocols for in vitro RNA synthesis ensuring the production of mg amount of pure and homogeneous molecules.

OB or Not OB: Idiosyncratic utilization of the tRNA-binding OB-fold domain in unicellular, pathogenic eukaryotes.

In this review, we examine the so-called OB-fold, a tRNA-binding domain homologous to the bacterial tRNA-binding protein Trbp111. We highlight the ability of OB-fold homologs to bind tRNA species and summarize their distribution in evolution. Nature has capitalized on the advantageous effects acquired when an OB-fold domain binds to tRNA by evolutionarily selecting this domain for fusion to different enzymes. Here, we review our current understanding of how the complexity of OB-fold-containing proteins and enzymes developed to expand their functions, especially in unicellular, pathogenic eukaryotes.

Aminoacylation of Plasmodium falciparum tRNA(Asn) and insights in the synthesis of asparagine repeats.

Genome sequencing revealed an extreme AT-rich genome and a profusion of asparagine repeats associated with low complexity regions (LCRs) in proteins of the malarial parasite Plasmodium falciparum. Despite their abundance, the function of these LCRs remains unclear. Because they occur in almost all families of plasmodial proteins, the occurrence of LCRs cannot be associated with any specific metabolic pathway; yet their accumulation must have given selective advantages to the parasite. Translation of these asparagine-rich LCRs demands extraordinarily high amounts of asparaginylated tRNA(Asn). However, unlike other organisms, Plasmodium codon bias is not correlated to tRNA gene copy number. Here, we studied tRNA(Asn) accumulation as well as the catalytic capacities of the asparaginyl-tRNA synthetase of the parasite in vitro. We observed that asparaginylation in this parasite can be considered standard, which is expected to limit the availability of asparaginylated tRNA(Asn) in the cell and, in turn, slow down the ribosomal translation rate when decoding asparagine repeats. This observation strengthens our earlier hypothesis considering that asparagine rich sequences act as "tRNA sponges" and help cotranslational folding of parasite proteins. However, it also raises many questions about the mechanistic aspects of the synthesis of asparagine repeats and about their implications in the global control of protein expression throughout Plasmodium life cycle.

Low Complexity Regions behave as tRNA sponges to help co-translational folding of plasmodial proteins.

In most organisms, the information necessary to specify the native 3D-structures of proteins is encoded in the corresponding mRNA sequences. Translational accuracy and efficiency are coupled and sequences that are slowly translated play an essential role in the concomitant folding of protein domains. Here, we suggest that the well-known mechanisms for the regulation of translational efficiency, which involves mRNA structure and/or asymmetric tRNA abundance, do not apply to all organisms. We propose that Plasmodium, the parasite responsible for malaria, uses an alternative strategy to slow down ribosomal speed and avoid multidomain protein misfolding during translation. In our model, the abundant Low Complexity Regions present in Plasmodium proteins replace the codon preferences, which influence the assembly of protein secondary structures.

Crystal growth of proteins, nucleic acids, and viruses in gels.

Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.

Microfluidic chips for the crystallization of biomacromolecules by counter-diffusion and on-chip crystal X-ray analysis.

Microfluidic devices were designed to perform on micromoles of biological macromolecules and viruses the search and the optimization of crystallization conditions by counter-diffusion, as well as the on-chip analysis of crystals by X-ray diffraction. Chips composed of microchannels were fabricated in poly-dimethylsiloxane (PDMS), poly-methyl-methacrylate (PMMA) and cyclo-olefin-copolymer (COC) by three distinct methods, namely replica casting, laser ablation and hot embossing. The geometry of the channels was chosen to ensure that crystallization occurs in a convection-free environment. The transparency of the materials is compatible with crystal growth monitoring by optical microscopy. The quality of the protein 3D structures derived from on-chip crystal analysis by X-ray diffraction using a synchrotron radiation was used to identify the most appropriate polymers. Altogether the results demonstrate that for a novel biomolecule, all steps from the initial search of crystallization conditions to X-ray diffraction data collection for 3D structure determination can be performed in a single chip.

Evidence for the existence in mRNAs of a hairpin element responsible for ribosome dependent pyrrolysine insertion into proteins.

In the methanogenic archae Methanosarcina barkeri, insertion of pyrrolysine, the 22nd amino acid, results from the decoding of an amber UAG codon in the mRNA of monomethylamine methyltransferases (MtmB). Sequence comparisons combined with structural enzymatic and chemical probing on M. barkeri MtmB1 mRNA demonstrate the presence of a hairpin motif located immediately after the redefined UAG codon. This structure of 86 nucleotides differs slightly from a proposal given in the literature and comprises four successive stems separated by three internal loops and closed by a large apical loop. Sequence alignments of MtmB mRNAs of different Methanosarcinacae reveal a conservation of the motif in both sequence and folding levels. The functional role of this motif as a signal leading to pyrrolysine insertion is discussed.

Atypical archaeal tRNA pyrrolysine transcript behaves towards EF-Tu as a typical elongator tRNA.

The newly discovered tRNA(Pyl) is involved in specific incorporation of pyrrolysine in the active site of methylamine methyltransferases in the archaeon Methanosarcina barkeri. In solution probing experiments, a transcript derived from tRNA(Pyl) displays a secondary fold slightly different from the canonical cloverleaf and interestingly similar to that of bovine mitochondrial tRNA(Ser)(uga). Aminoacylation of tRNA(Pyl) transcript by a typical class II synthetase, LysRS from yeast, was possible when its amber anticodon CUA was mutated into a lysine UUU anticodon. Hydrolysis protection assays show that lysylated tRNA(Pyl) can be recognized by bacterial elongation factor. This indicates that no antideterminant sequence is present in the body of the tRNA(Pyl) transcript to prevent it from interacting with EF-Tu, in contrast with the otherwise functionally similar tRNA(Sec) that mediates selenocysteine incorporation.