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International audience; Characterizing how multidrug-resistant bacteria circumvent the action of clinically used or novel antibiotics requires a detailed understanding of how the antibiotics interact with and cross bacterial membranes to accumulate in the cells and exert their action. When monitoring the interactions of drugs with bacteria it remains challenging to differentiate functionally relevant internalized drug levels from non-specific binding. Fluorescence is a method of choice for observing dynamics of biomolecules. In order to facilitate studies involving aminoglycoside antibiotics, we have generated fluorescently labeled aminoglycoside derivatives with uptake and bactericidal activities similar, albeit with a moderate loss, to those of the parent drug. The method combines fluorescence microscopy with fluorescence-activated cell sorting (FACS) using neomycin coupled to non-permeable cyanine dyes. Fluorescence imaging allowed membrane-bound antibiotic to be distinguished from molecules in the cytoplasm. Patterns of uptake were assigned to different populations in the FACS analysis. Our study illustrates how fluorescent derivatives of an aminoglycoside enable a robust characterization of the three components of uptake: membrane binding, EDPI, and EDPII. Because EDPI levels are weak compared to the two other types of accumulation and critical for the action of these drugs, the three components of uptake must be taken into account separately when drawing conclusions about aminoglycoside function.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the ongoing COVID-19 pandemic, like many other viruses, uses programmed ribosomal frameshifting (PRF) to enable synthesis of multiple proteins from its compact genome. In independent analyses, we evaluated the PRF regions of all SARS-CoV-2 sequences available in GenBank and from the Global Initiative on Sharing All Influenza Data for variations. Of the 5,156 and 27,153 sequences analyzed, respectively, the PRF regions were identical in 95.7% and 97.2% of isolates. The most common change from the reference sequence was from C to U at position 13,536, which lies in the three-stemmed pseudoknot known to stimulate frameshifting. With the conversion of the G 13493 -C 13536 Watson-Crick pair to G-U, the SARS-CoV-2 PRF closely resembles its counterpart in the Middle East respiratory syndrome coronavirus. The occurrence of this change increased from 0.5 to 3% during the period of March to May 2020.; The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the ongoing COVID-19 pandemic, like many other viruses, uses programmed ribosomal frameshifting (PRF) to enable synthesis of multiple proteins from its compact genome. In independent analyses, we evaluated the PRF regions of all SARS-CoV-2 sequences available in GenBank and from the Global Initiative on Sharing All Influenza Data for variations. Of the 5,156 and 27,153 sequences analyzed, respectively, the PRF regions were identical in 95.7% and 97.2% of isolates. The most common change from the reference sequence was from C to U at position 13,536, which lies in the three-stemmed pseudoknot known to stimulate frameshifting. With the conversion of the G 13493-C 13536 Watson-Crick pair to G-U, the SARS-CoV-2 PRF closely resembles its counterpart in the Middle East respiratory syndrome coronavirus. The occurrence of this change increased from 0.5 to 3% during the period of March to May 2020.

Thermus thermophilus is an extremely thermophilic eubacterium that produces various polyamines. Aminopropylagmatine ureohydrolase (SpeB) and SAM decarboxylase-like protein 1 (SpeD1) are involved in the biosynthesis of spermidine from arginine. Because long and branched polyamines in T. thermophilus are synthesized from spermidine, the speB and speD1 gene-deleted strains (ΔspeB and ΔspeD1, respectively) cannot synthesize long and branched polyamines. Although neither strain grew at high temperatures (75°C) in minimal medium, both strains survived at 80°C when they were cultured at 70°C until the mid-log phase and then shifted to 80°C. We therefore prepared the ΔspeB and ΔspeD1 cells using this culture method. Microscopic analysis showed that both strains can survive for 10 h after the temperature shift. Although the modification levels of 2'-O-methylguanosine at position 18, N

Thermus thermophilus is an extremely thermophilic eubacterium that produces various polyamines. Aminopropylagmatine ureohydrolase (SpeB) and SAM decarboxylase-like protein 1 (SpeD1) are involved in the biosynthesis of spermidine from arginine. Because long and branched polyamines in T. thermophilus are synthesized from spermidine, the speB and speD1 gene-deleted strains (ΔspeB and ΔspeD1, respectively) cannot synthesize long and branched polyamines. Although neither strain grew at high temperatures (\textgreater75°C) in minimal medium, both strains survived at 80°C when they were cultured at 70°C until the mid-log phase and then shifted to 80°C. We therefore prepared the ΔspeB and ΔspeD1 cells using this culture method. Microscopic analysis showed that both strains can survive for 10 h after the temperature shift. Although the modification levels of 2'-O-methylguanosine at position 18, N(7) -methylguanosine at position 46, 5-methyluridine at position 54 and N(1) -methyladenosine at position 58 in the class I tRNA from both strains were normal, amounts of tRNA(Tyr) , tRNA(His) , rRNAs and 70S ribosomes were decreased after the temperature shift. Furthermore, in vivo protein synthesis in both strains was completely lost 10 h after the temperature shift. Thus, long and branched polyamines are required for at least the maintenance of 70S ribosome and some tRNA species at high temperatures.

To progress in basic science and drug development, convenient methodology for detecting specific biological molecules and their interaction in living organism is in high demand. After more than 20 years of increasing research efforts, micro and nanotechnologies are now mature to propose a new class of miniature devices and principles enabling compartmentalized bioassays. Among them, this review proposes various examples that include array of electro-active microwells for highly parallel single cell analysis, cost-effective nanofluidic for DNA separation, parallel enzymatic reaction in 100 pL droplet and high-throughput platform for membrane proteins assays. The micro devices are presented with relevant experiments to foresee their future contribution to translational research and drug discovery.

International audience; Fluorogenic RNA aptamers provide a powerful tool for study of RNA analogous to green fluorescent protein for the study of proteins. Spinach and Broccoli are RNAs selected in vitro or in vivo respectively to bind to an exogenous chromophore. They can be genetically inserted into an RNA of interest for live-cell imaging. Spinach aptamer has been altered to increase thermal stability and stabilize the desired folding. How well these fluorogenic RNA aptamers behave when inserted into structured cellular RNAs and how aptamer properties might be affected remains poorly characterized. Here, we report a study of the performance of distinct RNA Spinach and Broccoli aptamer sequences in isolation or inserted into the small subunit of the bacterial ribosome. We found that the ribosomal context helped maintaining the yield of the folded Baby Spinach aptamer; other versions of Spinach did not perform well in the context of ribosomes. In vivo, two aptamers clearly stood out. Baby Spinach and Broccoli aptamers yielded fluorescence levels markedly superior to all previous Spinach sequences including the super-folder tRNA scaffolded tSpinach2. Overall, the results suggest the use of Broccoli and Baby Spinach aptamers for live cell imaging of structured RNAs.

International audience; TrmFO is a N(5) , N(10) -methylenetetrahydrofolate (CH2 THF)-/FAD-dependent tRNA methyltransferase, which synthesizes 5-methyluridine at position 54 (m(5) U54) in tRNA. Thermus thermophilus is an extreme-thermophilic eubacterium, which grows in a wide range of temperatures (50-83 °C). In T. thermophilus, modified nucleosides in tRNA and modification enzymes form a network, in which one modification regulates the degrees of other modifications and controls the flexibility of tRNA. To clarify the role of m(5) U54 and TrmFO in the network, we constructed the trmFO gene disruptant (∆trmFO) strain of T. thermophilus. Although this strain did not show any growth retardation at 70 °C, it showed a slow-growth phenotype at 50 °C. Nucleoside analysis showed increase in 2'-O-methylguanosine at position 18 and decrease in N(1) -methyladenosine at position 58 in the tRNA mixture from the ∆trmFO strain at 50 °C. These in vivo results were reproduced by in vitro experiments with purified enzymes. Thus, we concluded that the m(5) U54 modification have effects on the other modifications in tRNA through the network at 50 °C. (35) S incorporations into proteins showed that the protein synthesis activity of ∆trmFO strain was inferior to the wild-type strain at 50 °C, suggesting that the growth delay at 50 °C was caused by the inferior protein synthesis activity.

Translation machinery of mollicutes: economy is possible for proteins, not much for RNAs . Ribosome Structure and Function 2016

As more RNA molecules with important cellular functions are discovered, there is a strong need to characterize their structures, functions, and interactions. Chemical and enzymatic footprinting methods are used to map RNA secondary and tertiary structure, to monitor ligand interactions and conformational changes, and in the study of protein–RNA interactions. These methods provide data at single-nucleotide resolution that nicely complements the structural information available from X-ray diffraction, nuclear magnetic resonance spectroscopy (NMR), or cryo-electron microscopy. Footprinting methods also complement the dynamic information derived from single-molecule Forster resonance energy transfer. RNA footprinting tools have been used for decades, but we have recently seen spectacular advances, for instance, the use in combination with massive parallel sequencing techniques. Large libraries of RNA molecules (small or large in size) can now be probed in high-throughput manner when RNA footprinting methods are combined with fluorescent probe technologies and automation. In this article, after a brief historical overview, we summarize recent advances in RNA–protein footprinting methodologies that now integrate tools for massive parallel analysis. WIREs RNA 2011, 3:557–566. doi: 10.1002/wrna.1119 For further resources related to this article, please visit the WIREs website.

We present a novel method, implemented in the form of a microfluidic device, for arraying and analyzing large populations of single cells. The device contains a large array of electroactive microwells where manipulation and analysis of large population of cells are carried out. On the device, single cells can be actively trapped in the microwells by dielectrophoresis (DEP) and then lysed by electroporation (EP) for subsequent analysis of the confined cell lysates. The DEP force in the selected dimensions of the microwells could achieve efficient trapping in nearly all the microwells (95%) in less than three minutes. Moreover, the positions of the cells in the microwells are maintained even when unstable flow of liquid is applied. This makes it possible to exchange the DEP buffer to a solution that will be subsequently used for stimulating or analyzing the trapped cells. After closing the microwells, EP is conducted to lyse the trapped cells by applying short electric pulses. Tight enclosure is critical to prevent dilution, diffusion and cross contamination of the cell lysates. We demonstrated the feasibility of our approach with an enzymatic assay measuring the intracellular-galactosidase activity. The use of this method should greatly help analysis of large populations of cells at the single-cell level. Furthermore, the method offers rapidity in the trapping and analysis of multiple cell types in physiological conditions that will be important to ensure the relevance of single cell analyses.

Coordinated translocation of the tRNA-mRNA complex by the ribosome occurs in a precise, stepwise movement corresponding to a distance of three nucleotides along the mRNA. Frameshift suppressor tRNAs generally contain an extra nucleotide in the anticodon loop and they subvert the normal mechanisms used by the ribosome for frame maintenance. The mechanism by which suppressor tRNAs traverse the ribosome during translocation is poorly understood. Here, we demonstrate translocation of a tRNA by four nucleotides from the A site to the P site, and from the P site to the E site. We show that translocation of a punctuated mRNA is possible with an extra, unpaired nucleotide between codons. Interestingly, the NMR structure of the four nucleotide anticodon stem-loop reveals a conformation different from the canonical tRNA structure. Flexibility within the loop may allow conformational adjustment upon A site binding and for interacting with the four nucleotide codon in order to shift the mRNA reading frame.

Many pathogenic viruses use a programmed -1 translational frameshifting mechanism to regulate synthesis of their structural and enzymatic proteins. Frameshifting is vital for viral replication. A slippery sequence bound at the ribosomal A and P sites as well as a downstream stimulatory RNA structure are essential for frameshifting. Conflicting data have been reported concerning the structure of the downstream RNA signal in human immunodeficiency virus type 1 (HIV-1). Here, the solution structure of the HIV-1 frameshifting RNA signal was solved by heteronuclear NMR spectroscopy. This structure reveals a long hairpin fold with an internal three-nucleotide bulge. The internal loop introduces a bend between the lower and upper helical regions, a structural feature often seen in frameshifting pseudoknots. The NMR structure correlates with chemical probing data. The upper stem rich in conserved G-C Watson-Crick base-pairs is highly stable, whereas the bulge region and the lower stem are more flexible.

Yeast Rnt1p is a member of the double-stranded RNA (dsRNA) specific RNase III family of endoribonucleases involved in RNA processing and RNA interference (RNAi). Unlike other RNase III enzymes, which recognize a variety of RNA duplexes, Rnt1p cleaves specifically RNA stems capped with the conserved AGNN tetraloop. This unusual substrate specificity challenges the established dogma for substrate selection by RNase III and questions the dsRNA contribution to recognition by Rnt1p. Here we show that the dsRNA sequence adjacent to the tetraloop regulates Rnt1p cleavage by interfering with RNA binding. In context, sequences surrounding the cleavage site directly influence the cleavage efficiency. Introduction of sequences that stabilize the RNA helix enhanced binding while reducing the turnover rate indicating that, unlike the tetraloop, Rnt1p binding to the dsRNA helix may become rate-limiting. These results suggest that Rnt1p activity is strictly regulated by a combination of primary and tertiary structural elements allowing a substrate-specific binding and cleavage efficiency.

In prokaryotes, the recoding of a UGA stop codon as a selenocysteine codon requires a special elongation factor (EF) SelB and a stem-loop structure within the mRNA called a selenocysteine insertion sequence (SECIS). Here, we used NMR spectroscopy to determine the solution structure of the SECIS mRNA hairpin and characterized its interaction with the mRNA-binding domain of SelB. Our structural and biochemical data identified the conserved structural features important for binding to EF SelB within different SECIS RNA sequences. In the free SECIS mRNA structure, conserved nucleotides are strongly exposed for recognition by SelB. Binding of the C-terminal domain of SelB stabilizes the RNA secondary structure. In the protein–RNA complex, a Watson–Crick loop base-pair leaves a GpU sequence accessible for SelB recognition. This GpU sequence at the tip of the capping tetraloop and a bulge uracil five Watson–Crick base-pairs apart from the GpU are essential for interaction with SelB.

Mollicutes is a class of parasitic bacteria that have evolved from a common Firmicutes ancestor mostly by massive genome reduction. With genomes under 1 Mbp in size, most Mollicutes species retain the capacity to replicate and grow autonomously. The major goal of this work was to identify the minimal set of proteins that can sustain ribosome biogenesis and translation of the genetic code in these bacteria. Using the experimentally validated genes from the model bacteria Escherichia coli and Bacillus subtilis as input, genes encoding proteins of the core translation machinery were predicted in 39 distinct Mollicutes species, 33 of which are culturable. The set of 260 input genes encodes proteins involved in ribosome biogenesis, tRNA maturation and aminoacylation, as well as proteins cofactors required for mRNA translation and RNA decay. A core set of 104 of these proteins is found in all species analyzed. Genes encoding proteins involved in post-translational modifications of ribosomal proteins and translation cofactors, post-transcriptional modifications of t+rRNA, in ribosome assembly and RNA degradation are the most frequently lost. As expected, genes coding for aminoacyl-tRNA synthetases, ribosomal proteins and initiation, elongation and termination factors are the most persistent (i.e. conserved in a majority of genomes). Enzymes introducing nucleotides modifications in the anticodon loop of tRNA, in helix 44 of 16S rRNA and in helices 69 and 80 of 23S rRNA, all essential for decoding and facilitating peptidyl transfer, are maintained in all species. Reconstruction of genome evolution in Mollicutes revealed that, beside many gene losses, occasional gains by horizontal gene transfer also occurred. This analysis not only showed that slightly different solutions for preserving a functional, albeit minimal, protein synthetizing machinery have emerged in these successive rounds of reductive evolution but also has broad implications in guiding the reconstruction of a minimal cell by synthetic biology approaches., Author Summary In all cells, proteins are synthesized from the message encoded by mRNA using complex machineries involving many proteins and RNAs. In this process, named translation, the ribosome plays a central role. The elements involved in both ribosome biogenesis and its function are extremely conserved in all organisms from the simplest bacteria to mammalian cells. Most of the 260 known proteins involved in translation have been identified and studied in the bacteria Escherichia coli and Bacillus subtilis, two common cellular models in biology. However, comparative genomics has shown that the translation protein set can be much smaller. This is true for bacteria belonging to the class Mollicutes that are characterized by reduced genomes and hence considered as models for minimal cells. Using homology inference approach and expert analyses, we identified the translation apparatus proteins for 39 of these organisms. Although striking variations were found from one group of species to another, some Mollicutes species require half as many proteins as E. coli or B. subtilis. This analysis allowed us to determine a set of proteins necessary for translation in Mollicutes and define the translation apparatus that would be required in a cellular chassis mimicking a minimal bacterial cell.

Aminoglycoside antibiotics bind directly to 16S rRNA in the 30S subunit of bacterial ribosomes and decrease the fidelity of translation. Aminoglycoside antibiotics remain important therapeutic agents and represent the archetype for RNA-targeted antibiotics. This chapter presents an overview of the investigations of how aminoglycoside antibiotics bind to rRNA and the insights these studies have provided into the decoding process. Throughout the chapter, the term "aminoglycoside" will implicitly refer to this subclass. It is within this conserved decoding region RNA that aminoglycoside antibiotics bind. The chemical groups that are common among aminoglycoside antibiotics direct specific interaction with the RNA. The major sequence difference between all prokaryotic ribosomes and all eukaryotic ribosomes in the aminoglycoside binding site is an A1408-toG1408 change. However, only low-level resistance was observed to G418 and paromomycin, which are the most effective aminoglycosides against eukaryotic organisms. The major mechanism of aminoglycoside resistance is enzymatic modification of the drug. Molecular contacts between A1492 and A1493 and mRNA during decoding may also explain the miscoding induced by aminoglycoside antibiotics. The work on aminoglycoside antibiotics has revealed the details of how aminoglycosides bind to the ribosome, how resistance occurs, and how selectivity for prokaryotes is achieved. Only the combination of structure determination, biochemical, and biophysical approaches provides true insights into the workings of the ribosome. The use of a small oligonucleotide was essential for this work.

Interest in the gene expression levels of pluripotent stem cells has increased in order to precisely understand cellular differentiation. Here, we propose a method utilizing a large number of arrayed microchambers to quantitatively measure an intracellular fluorescence protein that is genetically inserted to monitor a pluripotency marker protein, Nanog, in pluripotent stem cells. Individual cells are isolated and lysed by inducing an electric potential on the cell membrane within the tightly enclosed microchambers. The microchambers have a size that is comparable to the target cells, making it possible to trap single cells and restrict the dilution of the cell lysate. The amount of intracellular fluorescence proteins in a single cell is precisely quantified inside the well-defined volume of each microchamber. Our method will be a useful tool for high-throughput and parallelized read-outs of gene expression levels in individual cells in a large population of cells.

Aminoglycoside antibiotics that bind to the ribosomal A site cause misreading of the genetic code and inhibit translocation. The clinically important aminoglycoside, gentamicin C, is a mixture of three components. Binding of each gentamicin component to the ribosome and to a model RNA oligonucleotide was studied biochemically and the structure of the RNA complexed to gentamicin C1a was solved using magnetic resonance nuclear spectroscopy. Gentamicin C1a binds in the major groove of the RNA. Rings I and II of gentamicin direct specific RNA-drug interactions. Ring III of gentamicin, which distinguishes this subclass of aminoglycosides, also directs specific RNA interactions with conserved base pairs. The structure leads to a general model for specific ribosome recognition by aminoglycoside antibiotics and a possible mechanism for translational inhibition and miscoding. This study provides a structural rationale for chemical synthesis of novel aminoglycosides.

Aminoglycoside antibiotics that bind to ribosomal RNA in the aminoacyl-tRNA site (A-site) cause misreading of the genetic code and inhibit translocation. We have recently solved the structure of an A-site RNA-paromomycin complex. The structure suggested that rings I and II, common to all aminoglycosides that bind to the A-site, are the minimum motif for specific ribosome binding to affect translation. This hypothesis was tested biochemically and with a detailed comparative NMR study of interaction of the aminoglycosides paromomycin, neomycin, ribostamycin, and neamine with the A-site RNA. Our NMR data show that rings I and II of neomycin-class aminoglycosides are sufficient to confer specificity to the binding of the antibiotics to the model A-site RNA. Neomycin, paromomycin, ribostamycin and neamine bind in the major groove of the A-site RNA in a unique binding pocket formed by non-canonical base pairs and a bulged nucleotide. Similar NMR properties of the RNA and the diverse antibiotics within the different complexes formed with neomycin, paromomycin, ribostamycin and neamine suggest similar structures for these complexes.

Current methodologies for arraying proteins using cell-free protein synthesis on a chip have spatial limitations that prevent reaching ultra-high density necessary for high throughput analysis. To circumvent this, we developed an on-chip method based on microcompartmentalization of protein synthesis. Proteins are synthesized in arrayed micrometer scale chambers from confined DNA template molecules. On-chip protein expression is highly efficient and the method can be used with a minimal amount of template i.e. single DNA molecules to perform digitalized cell-free protein synthesis (d-CFPS). A functionalized surface at the floor of the tightly sealed microchambers enables direct capture of expressed proteins. A density of 10⁴ spots per mm² was achieved, which represents a gain by more than 3 orders of magnitude over conventional methods. This technique of forming such densely arrayed small protein spots is the first step towards the development of a general method that would allow fabrication of ultra-high density protein arrays for high-throughput analysis.

The codon-anticodon interaction on the ribosome occurs in the A site of the 30 S subunit. Aminoglycoside antibiotics, which bind to ribosomal RNA in the A site, cause misreading of the genetic code and inhibit translocation. Biochemical studies and nuclear magnetic resonance spectroscopy were used to characterize the interaction between the aminoglycoside antibiotic paromomycin and a small model oligonucle otide that mimics the A site of Escherichia coli 16 S ribosomal RNA. Upon chemical modification, the RNA oligonucleotide exhibits an accessibility pattern similar to that of 16 S rRNA in the 30 S subunit. In addition, the oligonucleotide binds specifically aminoglycoside antibiotics. The anti biotic binding site forms an asymmetric internal loop, caused by non-canonical base-pairs. Nucleotides that are important for binding of paromomycin were identified by performing quantitative footprinting on oligonucleotide sequence variants and include the C1407·G1494 base-pair, and A·U base-pair at positions 1410/1490, and nucleotides A1408, A1493 and U1495. The asymmetry of the internal loop, which requires the presence of a nucleotide in position 1492, is also crucial for antibiotic binding. Introduction into the oligonucleotide of base changes that are known to confer aminoglycoside resistance in 16 S rRNA result in weaker binding of paromomycin to the oligonucleotide. Oligonucleotides homologous to eukaryotic rRNA sequences show reduced binding of paromomycin, suggesting a physical origin for the species-specific action of aminoglycosides.

A few aminoacyl-tRNA synthetases are characterized by their ability to tightly bind a zinc atom. In the case of Escherichia coli methionyl-tRNA synthetase, a peptide of 21 residues (138--163) having a stable 3-D structure in solution is responsible for zinc binding [Fourmy, D., Meinnel, T., Mechulam, Y., & Blanquet, S. (1993) J. Mol. Biol. 231, 1066--1077; Fourmy, D., Dardel, F., & Blanquet, S. (1993) J. Mol. Biol. 231, 1078--1089]. This peptide, which belongs to a region connecting the two halves of the nucleotide-binding domain of methionyl-tRNA synthetase, is likely to form a modular domain close to the active center of the enzyme. In this study, two residues of the zinc-binding module, Asp138 and Arg139, are shown to contribute to the stabilization of the transition state of the reaction leading to the activation of methionine. Moreover, another residue, Phe135, located at the surface of the zinc-binding domain, is found to possibly guide the tRNA acceptor stem toward the active site of the enzyme during catalysis. The available data indicate an important functional role for the zinc-binding module of methionyl-tRNA synthetase, as well as for other modules connecting conserved secondary structure elements in the aminoacyl-tRNA synthetase family. The relation between the occurrence of such variable peptide modules and the expression of both substrate specificity and catalytic efficiency is discussed.

Stochastic events in gene expression, protein synthesis, and metabolite synthesis or degradation lead to cellular heterogeneity essential to life. In a tissue as we see in organs, there is strong heterogeneity among the constituting cells critical to its function. Thus, there exists a strong demand to develop new micro/nanosystems that would enable us to conduct single-cell analysis. This field is rapidly growing, as exemplified below with recent emerging technologies that now reveal sensitive single-cell "omics" analysis. We describe in the review some of the most promising technologies that will certainly transform our view of biology in the near future.

Stochastic events in gene expression, protein synthesis, and metabolite synthesis or degradation lead to cellular heterogeneity essential to life. In a tissue as we see in organs, there is strong heterogeneity among the constituting cells critical to its function. Thus, there exists a strong demand to develop new micro/nanosystems that would enable us to conduct single-cell analysis. This field is rapidly growing, as exemplified below with recent emerging technologies that now reveal sensitive single-cell "omics" analysis. We describe in the review some of the most promising technologies that will certainly transform our view of biology in the near future.

This paper describes an integrated bio-reaction platform composed of silicon nanotweezers and open microfluidics for real time biomechanical assays. The silicon nanotweezers can sense slight biological modification of the trapped sample due to stable frequency response with high Q factor in liquid. The microfluidic device integrates active valves for controlling the biological medium. Biomolecular assay (DNA/Hind III enzymatic reaction kinetics), and cell contraction/relaxation dynamic assay illustrate the capabilities of this integrated platform that is a breakthrough capability for real time and routine biological tests.

Methyltransferase enzymes that use S-adenosylmethionine as a cofactor to catalyze 5-methyl uridine (m5U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, but are restricted to the Thermococcales and Nanoarchaeota groups amongst the Archaea. The RNA m5U methyltransferases appear to have arisen in Bacteria and were then dispersed by horizontal transfer of an rlmD-type gene to the Archaea and Eukaryota. The bacterium Escherichia coli has three gene paralogs and these encode the methyltransferases TrmA that targets m5U54 in tRNAs, RlmC (formerly RumB) that modifies m5U747 in 23S rRNA, and RlmD (formerly RumA) the archetypical enzyme that is specific for m5U1939 in 23S rRNA. The thermococcale archaeon Pyrococcus abyssi possesses two m5U methyltransferase paralogs, PAB0719 and PAB0760, with sequences most closely related to the bacterial RlmD. Surprisingly, however, neither of the two P. abyssi enzymes displays RlmD-like activity in vitro. PAB0719 acts in a TrmA-like manner to catalyze m5U54 methylation in P. abyssi tRNAs, and here we show that PAB0760 possesses RlmC-like activity and specifically methylates the nucleotide equivalent to U747 in P. abyssi 23S rRNA. The findings indicate that PAB0719 and PAB0760 originated as RlmD-type m5U methyltransferases and underwent changes in target specificity after their acquisition by a Thermococcales ancestor from a bacterial source.

Cys/His motifs, found in several nucleic acid binding proteins, generally correspond to sites for the binding of metal atoms. Such a motif, comprising four Cys residues, occurs in the subunits of Escherichia coli methionyl-tRNA synthetase, a dimeric enzyme known to bind two zinc atoms. In this study, each of the four cysteines in the cysteine cluster (region 145 to 161) of E. coli methionyl-tRNA synthetase were successively changed into an alanine. Either substitution is sufficient to destabilize the tight binding of the zinc ion. Moreover, a peptide having a sequence corresponding to that of the 138 to 163 region of methionyl-tRNA synthetase has been prepared. It strongly binds one zinc atom, even in the presence of ethylene diamine tetraacetate. These data establish that, in methionyl-tRNA synthetase, the Cys motif of region 145 to 161 is actually the binding site for zinc. In addition, the mutation of each cysteine modifies the parameters of the methionine activation reaction, and appears to change the structure of the enzyme, as probed by an increased sensitivity of the mutant enzymes to trypsin attack. The possible role of the zinc atom and of its chelating residues in the folding of the active centre of methionyl-tRNA synthetase is discussed.

This paper demonstrates the real-time sensing of enzymatic reactions on a DNA bundle trapped by silicon nanotweezers. The digestion of DNA in protein solution (Hind III) is monitored by tracking the change in frequency response of the immersed tweezers. This new direct biomechanical detection clears the way for systematic bio tests at the molecular level by an integrated MEMS device.

Ribosomal frameshifting on viral RNAs relies on the mechanical properties of structural elements, often pseudoknots and more rarely stem-loops, that are unfolded by the ribosome during translation. In human immunodeficiency virus (HIV)-1 type B a long hairpin containing a three-nucleotide bulge is responsible for efficient frameshifting. This three-nucleotide bulge separates the hairpin in two domains: an unstable lower stem followed by a GC-rich upper stem. Toeprinting and chemical probing assays suggest that a hairpin-like structure is retained when ribosomes, initially bound at the slippery sequence, were allowed multiple EF-G catalyzed translocation cycles. However, while the upper stem remains intact the lower stem readily melts. After the first, and single step of translocation of deacylated tRNA to the 30 S P site, movement of the mRNA stem-loop in the 5′ direction is halted, which is consistent with the notion that the downstream secondary structure resists unfolding. Mechanical stretching of the hairpin using optical tweezers only allows clear identification of unfolding of the upper stem at a force of 12.8 ± 1.0 pN. This suggests that the lower stem is unstable and may indeed readily unfold in the presence of a translocating ribosome.

Many viruses regulate translation of polycistronic mRNA using a −1 ribosomal frameshift induced by an RNA pseudoknot. When the ribosome encounters the pseudoknot barrier that resists unraveling, transient mRNA–tRNA dissociation at the decoding site, results in a shift of the reading frame. The eukaryotic frameshifting pseudoknot from the beet western yellow virus (BWYV) has been well characterized, both structurally and functionally. Here, we show that in order to obtain eukaryotic levels of frameshifting efficiencies using prokaryotic Escherichia coli ribosomes, which depend upon the structural integrity of the BWYV pseudoknot, it is necessary to shorten the mRNA spacer between the slippery sequence and the pseudoknot by 1 or 2 nucleotides (nt). Shortening of the spacer is likely to re-establish tension and/or ribosomal contacts that were otherwise lost with the smaller E. coli ribosomes. Chemical probing experiments for frameshifting and nonframeshifting BWYV constructs were performed to investigate the structural integrity of the pseudoknot confined locally at the mRNA entry site. These data, obtained in the pretranslocation state, show a compact overall pseudoknot structure, with changes in the conformation of nucleotides (i.e., increase in reactivity to chemical probes) that are first “hit” by the ribosomal helicase center. Interestingly, with the 1-nt shortened spacer, this increase of reactivity extends to a downstream nucleotide in the first base pair (bp) of stem 1, consistent with melting of this base pair. Thus, the 3 bp that will unfold upon translocation are different in both constructs with likely consequences on unfolding kinetics.

International audience; We present a study of the photophysical properties of the BODIPY-FL fluorescent dye at the single molecule scale using total internal reflexion fluorescence microscopy. We characterized the saturation intensity and the maximum number of fluorescence photons for single BODIPY-FL in a usual buffer. We then studied the influence of the buffer depletion in oxygen and the addition of redox agents on the brightness and the lifetime before photobleaching of the BODIPY-FL. The great improvement of these two factors in such a medium allows the use of BODIPY-FL in single-molecule biophysics experiments.

Comparison of the amino-acid sequences of several methionyl-tRNA synthetases indicates the occurrence of a few conserved motifs, having a possible functional significance. The role of one of these motifs, centered at position 300 in the E. coli enzyme sequence, was assayed by the use of site-directed mutagenesis. Substitution of the His 301 or Trp 305 residues by Ala resulted in a large decrease in methionine affinity, whereas the change of Val 298 into Ala had only a moderate effect. The catalytic rate of the enzyme was unimpaired by these substitutions. It is concluded that the above conserved amino-acid region is located at or close to the amino-acid binding pocket of methionyl-tRNA synthetase.

Udgivelsesdato: 2008-May-16 RlmA(II) methylates the N1-position of nucleotide G748 in hairpin 35 of 23 S rRNA. The resultant methyl group extends into the peptide channel of the 50 S ribosomal subunit and confers resistance to tylosin and other mycinosylated macrolide antibiotics. Methylation at G748 occurs in several groups of Gram-positive bacteria, including the tylosin-producer Streptomyces fradiae and the pathogen Streptococcus pneumoniae. Recombinant S. pneumoniae RlmA(II) was purified and shown to retain its activity and specificity in vitro when tested on unmethylated 23 S rRNA substrates. RlmA(II) makes multiple footprint contacts with nucleotides in stem-loops 33, 34 and 35, and does not interact elsewhere in the rRNA. Binding of RlmA(II) to the rRNA is dependent on the cofactor S-adenosylmethionine (or S-adenosylhomocysteine). RlmA(II) interacts with the same rRNA region as the orthologous enzyme RlmA(I) that methylates at nucleotide G745. Differences in nucleotide contacts within hairpin 35 indicate how the two methyltransferases recognize their distinct targets.

Udgivelsesdato: 2007-Sep-14 The methyltransferase RlmA(II) (formerly TlrB) is found in many Gram-positive bacteria, and methylates the N-1 position of nucleotide G748 within the loop of hairpin 35 in 23S rRNA. Methylation of the rRNA by RlmA(II) confers resistance to tylosin and other mycinosylated 16-membered ring macrolide antibiotics. We have previously solved the solution structure of hairpin 35 in the conformation that is recognized by the RlmA(II) methyltransferase from Streptococcus pneumoniae. It was shown that while essential recognition elements are located in hairpin 35, the interactions between RlmA(II) and hairpin 35 are insufficient on their own to support the methylation reaction. Here we use biochemical techniques in conjunction with heteronuclear/homonuclear nuclear magnetic resonance spectroscopy to define the RNA structures that are required for efficient methylation by RlmA(II). Progressive truncation of the rRNA substrate indicated that multiple contacts occur between RlmA(II) and nucleotides in stem-loops 33, 34 and 35. RlmA(II) appears to recognize its rRNA target through specific surface shape complementarity at the junction formed by these three helices. This means of recognition is highly similar to that of the orthologous Gram-negative methyltransferase, RlmA(I) (formerly RrmA), which also interacts with hairpin 35, but methylates at the adjacent nucleotide G745.

International audience; In bacteria, selenocysteine (the 21st amino acid) is incorporated into proteins via machinery that includes SelB, a specific translational elongation factor. SelB binds to an mRNA hairpin called the selenocysteine-insertion sequence (SECIS) and delivers selenocysteyl-tRNA(Sec) to the ribosomal A site. The minimum C-terminal fragment (residues 478-614) of Escherichia coli SelB (SelB-WH3/4) required for SECIS binding has been overexpressed and purified. This protein was crystallized in complex with 23 nucleotides of the SECIS hairpin at 294 K using the hanging-drop vapour-diffusion method. A data set was collected to 2.3 A resolution from a single crystal at 100 K using ESRF beamline BM-30. The crystal belongs to space group C2, with unit-cell parameters a = 103.50, b = 56.51, c = 48.41 A. The asymmetric unit contains one WH3/4-domain-RNA complex. The Matthews coefficient was calculated to be 3.37 A3 Da(-1) and the solvent content was estimated to be 67.4%.

Elongation factor SelB is responsible for co-translational incorporation of selenocysteine (Sec) into proteins. The UGA stop codon is recoded as a Sec codon in the presence of a downstream mRNA hairpin. In prokaryotes, in addition to the EF-Tu-like N-terminal domains, a C-terminal extension containing four tandem winged-helix motifs (WH1-4) recognizes the mRNA hairpin. The 2.3-A resolution crystal structure of the Escherichia coli WH3/4 domains bound to mRNA with mutagenesis data reveal that the two WH motifs use the same structural elements to bind RNA. The structure together with the 2.6-A resolution structure of the WH1-4 domains from Moorella thermoacetica bound to RNA revealed that a salt bridge connecting WH2 to WH3 modules is disrupted upon mRNA binding. The results provide a structural basis for the molecular switch that may allow communication between tRNA and mRNA binding sites and illustrate how RNA acts as an activator of the switch. The structures show that tandem WH motifs not only provide an excellent scaffold for RNA binding but can also have an active role in the function of protein-RNA complexes.

We present here the use of fluorescent methodologies for structural and functional studies of RNA in place of radioactivity. The methods are highly sensitive and quantitative with the use of an infrared fluorescence imaging system. IRD-700 and IRD-800 labels are used for fluorescence detection. Chemical probing methods are largely used for mapping RNA secondary structure and to monitor ligand interactions and conformational changes involving individual bases of RNA. The new fluorescent primer extension methodology allows simple and fast chemical probing of RNA with high sensitivity. IRD-700 and IRD-800 labeled primers can also be used to monitor protein–RNA interactions by fluorescent mobility shift assays. The speed and ease of these approaches are advantages over prior methods that used hazardous radioisotopes. Structural and biochemical investigations of RNA should benefit from the use of these fluorescent methodologies.

SummarySelenocysteine (Sec) is the “21st” amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SelB, which recognizes tRNASec via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SelB-C). SelB functions in multiple translational steps: binding to SECIS mRNA and tRNASec, delivery of tRNASec onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SelB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SelB-RNA interaction is sequence independent, possibly reflecting SelB-tRNA/-rRNA recognitions. Based on these data, the dynamic SelB-ribosome-mRNA-tRNA interactions will be discussed.

A phtalimido group can be readily introduced at the 5″‐position of N‐Boc ribostamycin 6 under Mitsunobu reaction conditions. However, in the case of N‐Boc neomycin B 4 a competitive bicyclization process occurred within the ring IV of the antibiotic, which led to compound 8, whose structure was fully elucidated by NMR spectroscopic methods. Accordingly, particular care must be exercised to achieve this chemical modification of the four‐ring antibiotic.

RNase III enzymes are a highly conserved family of proteins that specifically cleave double-stranded RNA (dsRNA). These proteins are involved in a variety of cellular functions, including the processing of many non-coding RNAs, mRNA decay, and RNA interference. In yeast Rnt1p, a dsRNA-binding domain (dsRBD) recognizes its substrate by interacting with stems capped with conserved AGNN tetraloops. The enzyme uses the tetraloop to cut 14nt to 16nt away into the stem in a ruler-like mechanism. The solution structure of Rnt1p dsRBD complexed to one of its small nucleolar (sno) RNA substrate revealed non-sequence-specific contacts with the sugar-phosphate backbone in the minor groove of the AGNN fold and the two non-conserved tetraloop nucleotides. Recently, a new form of Rnt1p substrates lacking the conserved AGNN sequence but instead harboring an AAGU tetraloop was found at the 5' end of snoRNA 48 precursor. Here, we report the solution structure of this hairpin capped with an AAGU tetraloop. Some of the stacking interactions and the position of the turn in the sugar-phosphate backbone are similar to the one observed in the AGNN loop structure; however, the AAGU sequence adopts a different conformation. The most striking difference was found at the 3' end of the loop where Rnt1p interacts with AGNN substrates. The last nucleotide is extruded from the AAGU tetraloop structure in contrast to the compact AGNN fold. The AAGU hairpin structure suggests that Rnt1p recognizes substrates with different tetraloop structures, indicating that the structural repertoire specifically recognized by Rnt1p is larger than previously anticipated.

In bacteria, the selenocysteine-specific elongation factor SelB is necessary for incorporation of selenocysteine, the 21st amino acid, into proteins by the ribosome. SelB binds to an mRNA hairpin formed by the selenocysteine-insertion sequence (SECIS) and delivers selenocysteyl-tRNA (Sec-tRNASec) at the ribosomal A site. The minimum fragment (residues 512-634) of Moorella thermoacetica SelB (SelB-M) required for mRNA binding has been overexpressed and purified. The complex of SelB-M with 23 nucleotides of the SECIS mRNA hairpin was crystallized at 293 K using the hanging-drop vapour-diffusion or oil-batch methods. The crystals diffract to 2.3 A resolution using SPring-8 BL41XU and belong to the space group P2(1)2(1)2, with unit-cell parameters a = 81.69, b = 169.58, c = 71.69 A.

In bacteria, incorporation of selenocysteine, the 21(st) amino acid, into proteins requires elongation factor SelB, which has the unusual property of binding to both transfer RNA (tRNA) and mRNA. SelB binds to an mRNA hairpin formed by the selenocysteine insertion sequence (SECIS) with extremely high specificity, the molecular basis of which has been unknown. We have determined the crystal structure of the mRNA-binding domain of SelB in complex with SECIS RNA at a resolution of 2.3 A. This is the first example of a complex between an RNA and a winged-helix (WH) domain, a motif found in many DNA-binding proteins and recently discovered in RNA-binding proteins. Notably, RNA binding does not induce a major conformational change in the WH motif. The structure reveals a new mode of RNA recognition with a geometry that allows the complex to wrap around the small ribosomal subunit.

The bacterial rRNA methyltransferase RlmAII (formerly TlrB) contributes to resistance against tylosin-like 16-membered ring macrolide antibiotics. RlmAII was originally discovered in the tylosin-producer Streptomyces fradiae, and members of this subclass of methyltransferases have subsequently been found in other Gram-positive bacteria, including Streptococcus pneumoniae. In all cases, RlmAII methylates 23S rRNA at nucleotide G748, which is situated in a stem–loop (hairpin 35) at the macrolide binding site of the ribosome. The conformation of hairpin 35 recognized by RlmAII is shown here by NMR spectroscopy to resemble the anticodon loop of tRNA. The loop folds independently of the rest of the 23S rRNA, and is stabilized by a non-canonical G–A pair and a U-turn motif, rendering G748 accessible. Binding of S.pneumoniae RlmAII induces changes in NMR signals at specific nucleotides that are involved in the methyltransferase–RNA interaction. The conformation of hairpin 35 that interacts with RlmAII is radically different from the structure this hairpin adopts within the 50S subunit. This indicates that the hairpin undergoes major structural rearrangement upon interaction with ribosomal proteins during 50S assembly.

Aminoglycoside antibiotics specifically interact with a variety of RNA sequences, and in particular with the decoding region of 16S ribosomal RNA in the aminoacyl tRNA acceptor site (A-site). Ring II of aminoglycosides (2-deoxystreptamine) is the most conserved element among aminoglycoside antibiotics that bind to the A-site. NMR structures of aminoglycoside-A-site RNA complexes suggested that the 2-deoxystreptamine core of aminoglycosides specifically recognizes (5')G-U(3') and potentially (5')G-G(3') or (5')U-G(3') steps in the major groove of RNA. Here, we show that isolated deoxystreptamine specifically interacts with G-U steps within the major groove of the A-site RNA. The bulge residue of A-site RNA is required to open the major groove for accommodation of deoxystreptamine. The chemical groups of deoxystreptamine presented to the RNA by the framework of the 6-carbon ring modulate RNA recognition.

Rnt1p, the yeast orthologue of RNase III, cleaves rRNAs, snRNAs and snoRNAs at a stem capped with conserved AGNN tetraloop. Here we show that 9 bp long stems ending with AGAA or AGUC tetraloops bind to Rnt1p and direct specific but sequence-independent RNA cleavage when provided with stems longer than 13 bp. The solution structures of these two tetraloops reveal a common fold for the terminal loop stabilized by non-canonical A–A or A–C pairs and extensive base stacking. The conserved nucleotides are stacked at the 5′ side of the loop, exposing their Watson–Crick and Hoogsteen faces for recognition by Rnt1p. These results indicate that yeast RNase III recognizes the fold of a conserved single-stranded tetraloop to direct specific dsRNA cleavage.

The ribosome is the molecular motor responsible for the protein synthesis within all cells. Ribosome motions along the messenger RNA (mRNA) to read the genetic code are asynchronous and occur along multiple kinetic paths. Consequently, observation and manipulation at the single macromolecule level is desirable to unravel the complex dynamics involved. In this communication, we present the study of translation kinetics of single ribosomes via the direct observation of fluorescent amino-acid incorporations.In order to study the kinetics of amino-acid incorporation inside the growing protein by the ribosome, we use a home-made total internal reflection single-molecule fluorescence microscope (TIRFM). The mRNA-ribosome complex is attached to a polyethylene glycol modified glass coverslip surface by a streptavidin-biotin linkage. The ribosome is labelled with a quantum dot (QD) in order to localize it on the surface while a specific amino acid (lysine) is marked with Bodipy-FL [1]. This fluorescent dye is small enough to enter the ribosomal channel thus leaving intact ribosomal activity. The protein synthesis is observed in real time as the labelled amino acids are incorporated into the polypeptidic chain by the co-localization of QD and Bodipy-FL fluorescence signals.We will discuss the future application of this technique to single-molecule observation of the translation process, proof reading or even protein folding.Reference[1] K. Perronet, P. Bouyer, N. Westbrook, N. Soler, D. Fourmy, and S. Yoshizawa. Journal of Luminescence, 127, 264, 2007.

Translational fidelity is established by ribosomal recognition of the codon-anticodon interaction within the aminoacyl–transfer RNA (tRNA) site (A site) of the ribosome. Experiments are presented that reveal possible contacts between 16 S ribosomal RNA and the codon-anticodon complex. N1 methylation of adenine at position 1492 (A1492) and A1493 interfered with A-site tRNA binding. Mutation of A1492 and A1493 to guanine or cytosine also impaired A-site tRNA binding. The deleterious effects of A1492G or A1493G (or both) mutations were compensated by 2′fluorine substitutions in the mRNA codon. The results suggest that the ribosome recognizes the codon-anticodon complex by adenine contacts to the messenger RNA backbone and provide a mechanism for molecular discrimination of correct versus incorrect codon-anticodon pairs.

International audience; Elongation factor SelB is responsible for cotranslational incorporation of the 21(st) amino acid selenocysteine (Sec) into proteins. UGA stop codon is recoded as a Sec codon in presence of a downstream mRNA hairpin. Prokaryotic SelB has EF-Tu-like Nterminal domains and a C-terminal extension containing four tandem winged-helix motifs (WH1-4). The C-terminal extension recognizes the mRNA hairpin. Crystal structures of the Escherichia coli WH3/4 domains and WH1-4 domains from Moorella thermoacetica each bound to mRNA were determined. These structures provide insights into how the molecular switch that may allow communication between tRNA and mRNA binding sites is formed and illustrate how RNA acts as an activator of the switch. The structures show that tandem WH motifs not only provide an excellent scaffold for RNA binding but can also have an active role in the function of protein-RNA complexes.