Biomedical subjects

Brice Felden

Publications and source records attributed to Brice Felden.

17 recordsLinked to original sources

PubMed2006

Scaffolding as an organizing principle in trans-translation. The roles of small protein B and ribosomal protein S1.

A eubacterial ribosome stalled on a defective mRNA can be released through a quality control mechanism referred to as trans-translation, which depends on the coordinating binding actions of transfer-messenger RNA, small protein B, and ribosome protein S1. By means of cryo-electron microscopy, we obtained a map of the complex composed of a stalled ribosome and small protein B, which appears near the decoding center. This result suggests that, when lacking a codon, the A-site on the small subunit is a target for small protein B. To investigate the role of S1 played in trans-translation, we obtained a cryo-electron microscopic map, including a stalled ribosome, transfer-messenger RNA, and small protein Bs but in the absence of S1. In this complex, several connections between the 30 S subunit and transfer-messenger RNA that appear in the +S1 complex are no longer found. We propose the unifying concept of scaffolding for the roles of small protein B and S1 in binding of transfer-messenger RNA to the ribosome during trans-translation, and we infer a pathway of sequential binding events in the initial phase of trans-translation.

Cryoelectron Microscopy↗

PubMed2006

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 x 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.

Binding Sites↗

PubMed2006

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.

Frameshifting, Ribosomal↗

PubMed2005

Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains.

Small RNA (sRNA) genes are expressed in all organisms, primarily as regulators of translation and message stability. We have developed comparative genomic approaches to identify sRNAs that are expressed by Staphylococcus aureus, the most common cause of hospital-acquired infections. This study represents an in-depth analysis of the RNome of a Gram-positive bacterium. A set of sRNAs candidates were identified in silico within intergenic regions, and their expression levels were monitored by using microarrays and confirmed by Northern blot hybridizations. Two sRNAs were also detected directly from purification and RNA sequence determination. In total, at least 12 sRNAs are expressed from the S. aureus genome, five from the core genome and seven from pathogenicity islands that confer virulence and antibiotic resistance. Three sRNAs are present in multiple (two to five) copies. For the sRNAs that are conserved throughout the bacterial phylogeny, their secondary structures were inferred by phylogenetic comparative methods. In vitro binding assays indicate that one sRNA encoded within a pathogenicity island is a trans-encoded antisense RNA regulating the expression of target genes at the posttranscriptional level. Some of these RNAs show large variations of expression among pathogenic strains, suggesting that they are involved in the regulation of staphylococcal virulence.

Base Pairing↗

PubMed2005

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

Stalled bacterial ribosomes are freed by transfer-messenger RNA (tmRNA). With the help of small protein B (SmpB), protein synthesis restarts and tmRNA adds a tag to the stalled protein for destruction. The conformation of a 347 nt long tmRNA from a thermophile and its interactions with SmpB were monitored using structural probes. The RNA is highly folded, including the reading frame, with <30% of unpaired residues. Footprints between SmpB and tmRNA are in the elbow of the tRNA domain, in some pseudoknots including one essential for function and in the lower part of the stem exiting the tRNA domain. The footprints outside the tRNA domain are scattered onto the tmRNA sequence, but form a cluster onto its tertiary structure derived from cryo-EM data. Some footprints flank the first triplet to be translated in tmRNA, suggesting that SmpB participates in the insertion of the tmRNA-encoded reading frame into the decoding center. To discriminate between a conformational rearrangement of tmRNA and independent binding sites, surface plasmon resonance was used and has identified three independent binding sites of SmpB on the RNA, including the site on the tRNA domain. Accordingly, SmpB is proposed to move on the tmRNA scaffold during trans-translation.

Base Sequence↗

PubMed2005

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

Trans-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.

Aminoglycosides↗

PubMed2005

tmRNA and associated ligands: a puzzling relationship.

Translation is an efficient and accurate mechanism, needing thorough systems of control-quality to ensure the correspondence between the information carried by the messenger RNA (mRNA) and the newly synthesized protein. Among them, trans-translation ensures delivering of stalled ribosomes when translation occurs on truncated mRNAs in bacteria, followed by the degradation of the incomplete nascent proteins. This process requires transfer-messenger RNA (tmRNA), an original molecule acting as both a tRNA and an mRNA. tmRNA first enters the decoding site of stuck ribosomes and, despite the lack of any codon-anticodon interaction, acts as a tRNA by transferring its alanine to the incomplete protein. Translation then switches to a small internal coding sequence (mRNA domain), which encodes a tag directing the incomplete protein towards degradation. Although playing a central role during trans-translation, tmRNA function depends on associated proteins. Genetic, biochemical and recent structural data are starting to unravel how the process takes place, by involving three main protein partners. Small protein B (SmpB) interacts with the tRNA-like domain (TLD) of tmRNA and is indispensable and specific to the process. Elongation factor Tu (EF-Tu) binds simultaneously the TLD and brings aminoacylated tmRNA to the ribosome, as for canonical tRNAs. Ribosomal protein S1 forms complexes with tmRNA, facilitating its recruitment by the stalled ribosomes. The chronology of events, however, is poorly understood and recent data shed light on the functions attributed to the proteins involved in trans-translation. This review focuses on the puzzling relationship that tmRNA has with these three protein ligands, putting forward trans-translation as a highly dynamical process.

Ligands↗

PubMed2004

A small bacterial RNA regulates a putative ABC transporter.

A small noncoding bacterial ribonucleic acid of 62-64 nucleotides, RydC, was identified in the genomes of Escherichia coli, Salmonella, and Shigella. In vivo, RydC binds to the RNA-binding protein Hfq, and it is unstable when Hfq is absent. Mobility assays reveal that complex formation between RydC and Hfq is specific, with an apparent binding constant of approximately 300 nm. Sequence alignments combined with structural probing demonstrate that RydC folds as a pseudoknot. Hfq binds the loops crossing the deep and shallow grooves of the pseudoknotted RNA and reorganizes its overall conformation. An interaction with a polycistronic mRNA, yejABEF, which encodes a putative ABC transporter, was detected by affinity purification of immobilized RNA-Hfq complexes. In vivo, the yejABEF operon is expressed on minimal medium. Remarkably, its expression is reduced when RydC is absent, and the operon is degraded when RydC expression is stimulated. This observation correlates with the growth defects associated with a stimulation of its expression in vivo, generating a thermosensitive phenotype that affects growth on minimal media supplemented with glycerol, maltose, or ribose. We conclude that RydC regulates the yejABEF-encoded ABC permease at the mRNA level. This small RNA may contribute to optimal adaptation of some Enterobacteria to environmental conditions.

ATP-Binding Cassette Transporters↗

PubMed2004

Ribosome rescue by tmRNA requires truncated mRNAs.

tmRNA targets ribosomes, stalled either on truncated mRNAs or on mRNAs with slowly read sense or stop codons, tags the newly synthesized peptide chains for degradation and allows for their release by a class-1 release factor. We have studied in vitro how the rate of trans-transfer of a peptide from the P-site tRNA to tmRNA and the efficiency by which tmRNA competes with peptide release factors depend on the length of the mRNA downstream from the P-site. We show that the rate and efficiency of tmRNA action decrease rapidly with increasing down stream length and approach zero when it exceeds 15 bases. We demonstrate that tmRNA action is strongly stimulated by RelE cleavage of mRNA in the A site. We conclude that tmRNA action in vivo must always be preceded by mRNA truncation, and suggest that cleavage of ribosome bound mRNAs is a common element in different bacterial stress responses.

Bacterial Physiological Phenomena↗

PubMed2004

Pre-binding of small protein B to a stalled ribosome triggers trans-translation.

To rescue stalled ribosomes, eubacteria employ a molecule, transfer messenger RNA (tmRNA), which functions both as a tRNA and as an mRNA. With the help of small protein B (SmpB), tmRNA restarts protein synthesis and adds by the trans-translation mechanism a peptide tag to the stalled protein to target it for destruction by cellular proteases. Here, the cellular location and expression of endogenous SmpB were monitored in vivo. We report that SmpB is associated with 70S ribosomes and not in the soluble fraction, independently of the presence of tmRNA. In vitro, SmpB that is pre-bound to a stalled ribosome can trigger initiation of trans-translation. Our results demonstrate the existence of a novel pathway for the entry of tmRNA to the ribosome and for the trans-transfer of a nascent peptide chain from peptidyl-tRNA to charged tmRNA.

Blotting, Northern↗

PubMed2004

Polyamine derivatives as selective RNaseA mimics.

Site-selective scission of ribonucleic acids (RNAs) has attracted considerable interest, since RNA is an intermediate in gene expression and the genetic material of many pathogenic viruses. Polyamine-imidazole conjugates for site-selective RNA scission, without free imidazole, were synthesized and tested on yeast phenylalanine transfer RNA. These molecules catalyze RNA hydrolysis non-randomly. Within the polyamine chain, the location of the imidazole residue, the numbers of nitrogen atoms and their relative distances have notable influence on cleavage selectivity. A norspermine derivative reduces the cleavage sites to a unique location, in the anticodon loop of the tRNA, in the absence of complementary sequence. Experimental results are consistent with a cooperative participation of an ammonium group of the polyamine moiety, in addition to it's binding to the negatively charged ribose-phosphate backbone, as proton source, and the imidazole moiety as a base. There is correlation between the location of the magnesium binding sites and the RNA cleavage sites, suggesting that the protonated nitrogens of the polycationic chain compete with some of the magnesium ions for RNA binding. Therefore, the cleavage pattern is specific of the RNA structure. These compounds cleave at physiological pH, representing novel reactive groups for antisense oligonucleotide derivatives or to enhance ribozyme activity.

Anticodon↗

PubMed2003

Intergenic sequence inspector: searching and identifying bacterial RNAs.

Intergenic Sequence Inspector (ISI) is a program that helps in identifying bacterial ribonucleic acids (RNAs). It automatically extracts, selects and visualizes candidate intergenic regions (IGRs) that bear conservations between phylogenetically related species, displaying sequence and structural signatures of RNA genes encompassing putative promoters, terminators, RNA secondary structure predictions and the G+C percent of the selected sequences. ISI is intentionally modular to analyze various bacterial genomes according to their intrinsic specificities and to the available data. ISI provides a sum of information to the user who can evaluate whether or not an IGR is susceptible to express RNAs.

DNA, Intergenic↗

PubMed2003

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.

Alanine↗

PubMed2003

Crystal structure of the transfer-RNA domain of transfer-messenger RNA in complex with SmpB.

Accurate translation of genetic information into protein sequence depends on complete messenger RNA molecules. Truncated mRNAs cause synthesis of defective proteins, and arrest ribosomes at the end of their incomplete message. In bacteria, a hybrid RNA molecule that combines the functions of both transfer and messenger RNAs (called tmRNA) rescues stalled ribosomes, and targets aberrant, partially synthesized, proteins for proteolytic degradation. Here we report the 3.2-A-resolution structure of the tRNA-like domain of tmRNA (tmRNA(Delta)) in complex with small protein B (SmpB), a protein essential for biological functions of tmRNA. We find that the flexible RNA molecule adopts an open L-shaped conformation and SmpB binds to its elbow region, stabilizing the single-stranded D-loop in an extended conformation. The most striking feature of the structure of tmRNA(Delta) is a 90 degrees rotation of the TPsiC-arm around the helical axis. Owing to this unusual conformation, the SmpB-tmRNA(Delta) complex positioned into the A-site of the ribosome orients SmpB towards the small ribosomal subunit, and directs tmRNA towards the elongation-factor binding region of the ribosome. On the basis of this structure, we propose a model for the binding of tmRNA on the ribosome.

Bacterial Proteins↗

PubMed2003

Inhibition of transfer messenger RNA aminoacylation and trans-translation by aminoglycoside antibiotics.

Transfer messenger RNA (tmRNA) directs the modification of proteins of which the biosynthesis has stalled or has been interrupted. Here, we report that aminoglycosides can interfere with this quality control system in bacteria, termed trans-translation. Neomycin B is the strongest inhibitor of tmRNA aminoacylation with alanine (K(i) value of approximately 35 micro m), an essential step during trans-translation. The binding sites of neomycin B do not overlap with the identity determinants for alanylation, but the aminoglycoside perturbs the conformation of the acceptor stem that contains the aminoacylation signals. Aminoglycosides reduce the conformational freedom of the transfer RNA-like domain of tmRNA. Additional contacts between aminoglycosides and tmRNA are within the tag reading frame, probably also disturbing reprogramming of the stalled ribosomes prior protein tagging. Aminoglycosides impair tmRNA aminoacylation in the presence of all of the transfer RNAs from Escherichia coli, small protein B, and elongation factor Tu, but when both proteins are present, the inhibition constant is 1 order of magnitude higher. SmpB and elongation factor Tu have RNA chaperone activities, ensuring that tmRNA adopts an optimal conformation during aminoacylation.

Acylation↗

PubMed2002

Two-piece tmRNA in cyanobacteria and its structural analysis.

tmRNA acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis. The permuted gene structure found in a group of cyanobacteria is shown to produce a two-piece mature tmRNA, as had been observed previously for the independently permuted gene of alpha-proteobacteria. The pieces have been mapped onto the gene sequence and aligned for the permuted cyanobacterial tmRNA sequences, including four novel sequences. Structural probing and base pair co-variations support a secondary structure model in which two pairings in the tRNA-like domain hold the two pieces together, and the coding piece bearing the tag reading frame additionally contains a single transient pseudoknot and three other stem-loops. This represents a dramatic reduction in pseudoknot number from the five present in one-piece cyanobacterial tmRNA.

Base Sequence↗

PubMed2002

Ribosomal protein S1 induces a conformational change of tmRNA; more than one protein S1 per molecule of tmRNA.

tmRNA (10Sa RNA, ssrA) acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis. Ribosomal protein S1 is required for tmRNA binding to isolated and poly U-programmed ribosomes. Mobility assays on native gels indicate that the binding curves of both recombinant and purified proteins S1 from E. coli is biphasic with apparent binding constants of approximately 90 and approximately 300 nM, respectively, suggesting that more than one protein interacts with tmRNA. Structural probing of native tmRNA in the presence and absence of the purified protein suggest that when S1 binds, tmRNA undergoes a significant conformational change. In the presence of the protein, nucleotides from tmRNA with enhanced (H2, H3, PK1, PK2, PK4, in and around the first triplet to be translated), or decreased (H5 and PK2), reactivity towards a probe specific for RNA single-strands are scattered throughout the molecule, with the exception of the tRNA-like domain that may be dispensable for the interaction. Converging experimental evidence suggests that ribosomal protein S1 binds to pseudoknot PK2. Previous structural studies of tmRNA in solution have revealed several discrepancies between the probing data and the phylogeny, and most of these are reconciled when analyzing tmRNA structure in complex with the protein(s). Ribosomal protein(s) S1 is proposed to set tmRNA in the mRNA mode, relieving strains that may develop when translating a looped mRNA.

Autoradiography↗