Small protein B interacts with the large and the small subunits of a stalled ribosome during trans-translation

During trans-translation, stalled bacterial ribosomes are rescued by small protein B (SmpB) and by transfer-messenger RNA (tmRNA). Stalled ribosomes switch translation from the defective messages to a short internal reading frame on tmRNA that tags the nascent peptide chain for degradation and recycles the ribosomes. We present evidences that SmpB binds the large and small ribosomal subunits in vivo and in vitro. The binding between SmpB and the ribosomal subunits is very tight, with a dissociation constant of 1.7 × 10(−10) M, similar to its K(D) for the 70S ribosome or for tmRNA. tmRNA displaces SmpB from its 50S binding but not from the 30S. In vivo, SmpB is detected on the 50S when trans-translation is impaired by lacking tmRNA or a functional SmpB. SmpB contacts the large subunit transiently and early during the trans-translational process. The affinity of SmpB for the two ribosomal subunits is modulated by tmRNA in the course of trans-translation. It is the first example of two copies of the same protein interacting with two different functional sites of the ribosomes

Interaction of a Dimeric Single-Stranded DNA-Binding Protein (G5P) with DNA Hairpins. A Molecular Beacon Study

Gene-V protein (G5P/GVP) is a single-stranded (ss)DNA-binding protein (SBP) of bacteriophage f1 that is required for DNA synthesis and repair. In solution, it exists as a dimer that binds two antiparallel ssDNA strands with high affinity in a cooperative manner, forming a left-handed helical protein–DNA filament. Here, we report on fluorescence studies of the interaction of G5P with different DNA oligonucleotides having a hairpin structure (molecular beacon, MB) with a seven base-pair stem (dT24-stem7, dT18-stem7), as well as with DNA oligonucleotides (dT38, dT24) without a defined secondary structure. All oligonucleotides were end-labeled with a Cy3-fluorophore and a BHQ2-quencher. In the case of DNA oligonucleotides without a secondary structure, an almost complete quenching of their strong fluorescence (with about 5% residual intensity) was observed upon the binding of G5P. This implies an exact alignment of the ends of the DNA strand(s) in the saturated complex. The interaction of the DNA hairpins with G5P led to the unzipping of the base-paired stem, as revealed by fluorescence measurements, fluorescence microfluidic mixing experiments, and electrophoretic mobility shift assay data. Importantly, the disruption of ssDNA’s secondary structure agrees with the behavior of other single-stranded DNA-binding proteins (SBPs). In addition, substantial protein-induced fluorescence enhancement (PIFE) of the Cy3-fluorescence was observed

Les mécanismes de résistance aux antibiotiques chez Staphylococcus Aureus

RENNES1-BU Santé (352382103) / SudocSudocFranceF

The highest affinity binding site of small protein B on transfer messenger RNA is outside the tRNA domain

Eubacterial ribosomes stalled on defective mRNAs are released through a mechanism referred to as trans-translation, depending on the coordinated actions of small protein B (SmpB) and transfer messenger RNA (tmRNA). A series of tmRNA variants with deletions in each structural domain were produced. Their structures were monitored by enzymatic and chemical probes in vitro, in the presence and absence of SmpB. Dissociation constants between these RNAs and SmpB from Aquifex aeolicus were derived by surface plasmon resonance (SPR) combined with filter binding assays. Three independent experimental evidences, including filter binding assays, SPR, and concentration titrations of the RNA–protein reactivity changes toward structural probes, indicate that the binding site that has the highest affinity for the protein is located outside the tRNA domain, upstream of the internal tag. The minimal tmRNA fragment that contains this high affinity site for SmpB, and also contains another site of lower affinity, includes the tag reading frame and three downstream pseudoknots that form a ring structure in solution

Emerging Functions for the Staphylococcus aureus RNome.

International audienceStaphylococcus aureus is a leading pathogen for animals and humans, not only being one of the most frequently isolated bacteria in hospital-associated infections but also causing diseases in the community. To coordinate the expression of its numerous virulence genes for growth and survival, S. aureus uses various signalling pathways that include two-component regulatory systems, transcription factors, and also around 250 regulatory RNAs. Biological roles have only been determined for a handful of these sRNAs, including cis, trans, and cis-trans acting RNAs, some internally encoding small, functional peptides and others possessing dual or multiple functions. Here we put forward an inventory of these fascinating sRNAs; the proteins involved in their activities; and those involved in stress response, metabolisms, and virulence

Independent binding sites of small protein B onto transfer-messenger RNA during trans-translation

during trans-translatio

tmRNA decreases the bactericidal activity of aminoglycosides and the susceptibility to inhibitors of cell wall synthesis.

International audienceTrans-translation is a process that recycles ribosomes stalled on problematic mRNAs. tmRNA, coded by the DeltassrA gene, is a major component of trans-translation. Bacteria lacking tmRNA are more sensitive to several inhibitors of protein synthesis when compared to a wild type strain. We measured bacterial growth of the DeltassrA and wild type strains in Escherichia coli in the presence of 14 antibiotics including some that do not target protein synthesis. Both the optical density of the bacterial cultures and the number of viable cells were monitored. For the ribosome-targeted antibiotics, sensitization was observed on erythromycin, chloramphenicol, kanamycin, puromycin and streptomycin. Minor or no effects were observed with clindamycin, tetracycline and spectinomycin. Surprisingly, the DeltassrA strain is more sensitive than wild type to inhibitors of cell wall synthesis: fosfomycin and ampicillin. No growth difference was observed on drugs with other target sites: ofloxacin, norfloxacin, rifampicin and trimethoprim. Sensitization to antibiotics having target sites other than the ribosome suggests that trans-translation could influence antibiotic-induced stress responses. In trans-translation-deficient bacteria, cell death is significantly enhanced by the two aminoglycosides that induce translational misreading, streptomycin and kanamycin

sRNAs from the <i>S. aureus</i> RNome implicated in bacterial virulence.

<p>Multitasking RNAIII is the effector of quorum sensing to perceive bacterial population density and regulates multiple targets involved in peptidoglycan metabolism, adhesion, exotoxins production, and virulence. RNAIII internally encodes hemolysin δ (blue). RNAIII contains at least three repressor domains (red) containing accessible UCCC motifs that interact, by antisense pairings, with the ribosome binding sites of numerous target mRNAs for translational repression (Tr.R), some triggering endoribonuclease III (RNase III) cleavages to induce target mRNA degradations and irreversible gene expression decay. Translation of at least two exotoxins is activated by RNAIII, one encoded (hlδ), and another (hlα) by translation activation (Tr.A). SprD is expressed from the genome of a converting phage and interacts, by antisense pairings, with the 5′ part of the <i>sbi</i> mRNA encoding an immune evasion molecule. SprD possesses an important role in <i>S. aureus</i> virulence, but the mechanism of its control is yet to be elucidated, with Sbi being only one player among others. The 891-nucleotide long SSR42 affects extracellular virulence expression, hemolysis, neutrophil virulence, and pathogenesis and contains a putative internal ORF. The mechanisms of target regulation remain to be elucidated. The SCCmec-encoded <i>psm-mec</i> RNA suppresses <i>agrA</i> translation and attenuates MRSA virulence, acting as a dual-function RNA regulator.</p

RsaE controls central metabolic pathways.

<p>RsaE regulates, directly or indirectly, the expression of several genes involved in amino acid synthesis, peptides transport, carbohydrate metabolism, and the TCA cycle. RsaE directly regulates the TCA cycle by inhibiting <i>sucD</i> mRNA translation coding for one of the subunits of the succinyl-Coa synthase. It alters the purine biosynthetic pathway via the down-regulation of some enzymes involved in the folate-dependent, one-carbon metabolism. RsaE uses multiple binding sites for the regulation of the <i>opp3BCDFA</i> mRNA expressing an oligopeptide transporter involved in nutrient transport. RsaE pairs directly with sites overlapping the ribosome binding site of the upstream (<i>opp3B</i>) and distal (<i>opp3A</i>) genes from the operon to inhibit their translations. RsaE modulates the intracellular pool of amino acid by down-regulating the expression of an oligopeptide transporter and by up-regulating genes that produce amino acid synthesis enzymes. In some <i>S. aureus</i> strains, RsaE expression is controlled by the <i>agr</i> quorum-sensing system in response to autoinducing peptide (AIP), and it depends on the σ^B regulon. The plain and dashed lines indicate the direct and indirect gene regulations, respectively (red bars: inhibitions, black arrows: stimulations).</p