Recognition of tRNA with a UAU anticodon by isoleucyl-tRNA synthetase in lactic acid bacteria.

In almost all eubacteria, the AUA codon is translated by tRNA bearing lysidine at the wobble position. Lysidine is introduced by tRNA lysidine synthetase (TilS) via post-transcriptional modification of the cytidine of tRNA (CAU). Lactobacillus casei and Lactobacillus plantarum have tilS homologues and tRNA (CAU) genes.

Fluorescent Aminoglycoside Antibiotics and Methods for Accurately Monitoring Uptake by Bacteria.

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 nonspecific binding. Fluorescence is a method of choice for observing dynamics of biomolecules.

Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry.

A method based on supercritical fluid chromatography coupled to high resolution mass spectrometry for the profiling of canonical and modified nucleosides was optimized, and compared to classical reverse-phase liquid chromatography in terms of separation, number of detected modified nucleosides and sensitivity. Limits of detection and quantification were measured using statistical method and quantifications of twelve nucleosides of a tRNA digest from E. coli are in good agreement with previously reported data.

Characterization of redundant tRNAIles with CAU and UAU anticodons in Lactobacillus plantarum.

In most eubacteria, the minor AUA isoleucine codon is decoded by tRNAIle2, which has a lysidine (L) in the anticodon loop. The lysidine is introduced by tRNAIle-lysidine synthetase (TilS) through post-transcriptional modification of cytidine to yield an LAU anticodon. Some bacteria, Lactobacillus plantarum for example, possess two tRNAIle2(UAU) genes in addition to, two tRNAIle2(CAU) genes and the tilS gene.

DNA-Guided Delivery of Single Molecules into Zero-Mode Waveguides.

Zero-mode waveguides (ZMWs) are powerful analytical tools corresponding to optical nanostructures fabricated in a thin metallic film capable of confining an excitation volume to the range of attoliters. This small volume of confinement allows single-molecule fluorescence experiments to be performed at physiologically relevant concentrations of fluorescently labeled biomolecules. Exactly one molecule to be studied must be attached at the floor of the ZMW for signal detection and analysis; however, the massive parallelism of these nanoarrays suffers from a Poissonian-limited distribution of these biomolecules.

Long and branched polyamines are required for maintenance of the ribosome, tRNA and tRNA in Thermus thermophilus cells at high temperatures.

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.

Use of Baby Spinach and Broccoli for imaging of structured cellular RNAs.

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.

Folate-/FAD-dependent tRNA methyltransferase from Thermus thermophilus regulates other modifications in tRNA at low temperatures.

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.

Predicting the minimal translation apparatus: lessons from the reductive evolution of mollicutes.

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.

Ultra-high density protein spots achieved by on chip digitalized protein synthesis.

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.

Enzymatic reaction in droplets manipulated with liquid dielectrophoresis.

Droplet generation and transportation for biological reactions are conducted with liquid dielectrophoresis (LDEP), forming two hundred picoliter droplets and aligning them in an open environment above the micro-machined electrodes. The generation of the dielectrophoresis signals was critically examined to actuate droplets in biological solutions without excessive Joule heating. Enzymatic reactions between β-galactosidase and fluorescein di-β-D-galactopyranoside were succeeded in manipulated droplets, which was confirmed by fluorescence imaging.

Protein-RNA footprinting: an evolving tool.

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.

Micro and nanotechnological tools for study of RNA.

Micro and nanotechnologies have originally contributed to engineering, especially in electronics. These technologies enable fabrication and assembly of materials at micrometer and nanometer scales and the manipulation of nano-objects. The power of these technologies has now been exploited in analyzes of biologically relevant molecules.

Complex transcriptional regulation of citrate metabolism in Clostridium perfringens.

A Gram-positive, spore-forming bacterium, Clostridium perfringens, possesses genes for citrate metabolism, which might play an important role in the utilization of citrate as a sole carbon source. In this study, we identified a chromosomal citCDEFX-mae-citS operon in C. perfringens strain 13, which is transcribed on three mRNAs of different sizes.

Rapid detection of a cocaine-binding aptamer using biological nanopores on a chip.

This paper describes a methodology for the rapid and highly selective detection of cocaine using a membrane protein channel combined with a DNA aptamer. The DNA aptamer recognizes the cocaine molecule with high selectivity. We successfully detected a low concentration of cocaine (300 ng/mL, the drug test cutoff limit) within 60 s using a biological nanopore embedded in a microchip.

Lipid-coated microdroplet array for in vitro protein synthesis.

Monitoring complex biological assays such as in vitro protein synthesis over long periods in micrometer-sized cavities of poly(dimethyl siloxane) (PDMS) microfluidic devices requires a strategy that solves the adsorption and absorption problems on PDMS surfaces. In this study, we developed a technique that instantaneously arrays aqueous microdroplets coated with a phospholipid membrane within a single microfluidic device. The simple lipid bilayer coating effectively inhibits the adsorption of proteins and DNA, whereas the encapsulation of the droplet reduces the area in contact with the PDMS surface, resulting in decreased absorption in part.

Identification of a two-component VirR/VirS regulon in Clostridium perfringens.

Clostridium perfringens, a Gram-positive anaerobic pathogen, is a causative agent of human gas gangrene that leads to severe rapid tissue destruction and can cause death within hours unless treated immediately. Production of several toxins is known to be controlled by the two-component VirR/VirS system involving a regulatory RNA (VR-RNA) in C. perfringens.

Interaction of the HIV-1 frameshift signal with the ribosome.

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.

Footprinting analysis of BWYV pseudoknot-ribosome complexes.

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.

The many levels of control on bacterial selenoprotein synthesis.

Selenium shares many chemical facets with sulphur but differs from it in the redox potential, especially of the Se(2-)/S(2-) oxidation state. The higher chemical reactivity of the deprotonated selenol has been used by Biology in the synthesis of the amino acid selenocysteine and its DNA-encoded incorporation into specific positions of proteins to enhance their structural role or their activity. Since selenocysteine is a steric isomer of cysteine, numerous control mechanisms have been developed which prevent cross-intrusion of the elements during biosynthesis and insertion.

Interaction of the tylosin-resistance methyltransferase RlmA II at its rRNA target differs from the orthologue RlmA I.

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.

Molecular switch in tandem winged-helix motifs of elongation factor SelB.

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 N-terminal domains and a C-terminal extension containing four tandem winged-helix motifs (WH1-4).