RESUMEN
For energy-efficient magnetic memories, switching of perpendicular magnetization by spin-orbit torque (SOT) appears to be a promising solution. This SOT switching requires the assistance of an in-plane magnetic field to break the symmetry. Here, we demonstrate the field-free SOT switching of a perpendicularly magnetized thulium iron garnet (Tm3Fe5O12, TmIG). The polarity of the switching loops, clockwise or counterclockwise, is determined by the direction of the initial current pulses, in contrast with field-assisted switching where the polarity is controlled by the direction of the magnetic field. From Brillouin light scattering, we determined the Dzyaloshinskii-Moriya interaction (DMI) induced by the Pt-TmIG interface. We will discuss the possible origins of field-free switching and the roles of the interfacial DMI and cubic magnetic anisotropy of TmIG. This discussion is substantiated by magnetotransport, Kerr microscopy, and micromagnetic simulations. Our observation of field-free electrical switching of a magnetic insulator is an important milestone for low-power spintronic devices.
Asunto(s)
Citrobacter/genética , Tracto Gastrointestinal/microbiología , Transferencia de Gen Horizontal , Klebsiella pneumoniae/genética , beta-Lactamasas/genética , Adolescente , Antibacterianos/farmacología , Citrobacter/efectos de los fármacos , Citrobacter/aislamiento & purificación , Humanos , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/aislamiento & purificación , Masculino , Pruebas de Sensibilidad Microbiana , Resistencia betalactámicaRESUMEN
IS911 has provided a powerful model for studying the transposition of members of a large class of transposable element: the IS3 family of bacterial Insertion Sequences (IS). These transpose by a Copy-out-Paste-in mechanism in which a double-strand IS circle transposition intermediate is generated from the donor site by replication and proceeds to integrate into a suitable double strand DNA target. This is perhaps one of the most common transposition mechanisms known to date. Copy-out-Paste-in transposition has been adopted by members of at least eight large IS families. This chapter details the different steps of the Copy-out-Paste-in mechanism involved in IS911 transposition. At a more biological level it also describes various aspects of regulation of the transposition process. These include transposase production by programmed translational frameshifting, transposase expression from the circular intermediate using a specialized promoter assembled at the circle junction and binding of the nascent transposase while it remains attached to the ribosome during translation (co-translational binding). This co-translational binding of the transposase to neighboring IS ends provides an explanation for the longstanding observation that transposases show a cis-preference for their activities.
Asunto(s)
Bacterias/genética , Elementos Transponibles de ADN , Bacterias/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Recombinación Genética , Transposasas/genética , Transposasas/metabolismoRESUMEN
Programmed ribosomal -1 frameshifting is a non-standard decoding process occurring when ribosomes encounter a signal embedded in the mRNA of certain eukaryotic and prokaryotic genes. This signal has a mandatory component, the frameshift motif: it is either a Z_ZZN tetramer or a X_XXZ_ZZN heptamer (where ZZZ and XXX are three identical nucleotides) allowing cognate or near-cognate repairing to the -1 frame of the A site or A and P sites tRNAs. Depending on the signal, the frameshifting frequency can vary over a wide range, from less than 1% to more than 50%. The present study combines experimental and bioinformatics approaches to carry out (i) a systematic analysis of the frameshift propensity of all possible motifs (16 Z_ZZN tetramers and 64 X_XXZ_ZZN heptamers) in Escherichia coli and (ii) the identification of genes potentially using this mode of expression amongst 36 Enterobacteriaceae genomes. While motif efficiency varies widely, a major distinctive rule of bacterial -1 frameshifting is that the most efficient motifs are those allowing cognate re-pairing of the A site tRNA from ZZN to ZZZ. The outcome of the genomic search is a set of 69 gene clusters, 59 of which constitute new candidates for functional utilization of -1 frameshifting.
Asunto(s)
Escherichia coli/genética , Sistema de Lectura Ribosómico , ARN Mensajero/química , Genes Bacterianos , Motivos de Nucleótidos , Nucleótidos/análisisRESUMEN
Bacterial genome annotations contain a number of coding sequences (CDSs) that, in spite of reading frame disruptions, encode a single continuous polypeptide. Such disruptions have different origins: sequencing errors, frameshift, or stop codon mutations, as well as instances of utilization of nontriplet decoding. We have extracted over 1,000 CDSs with annotated disruptions and found that about 75% of them can be clustered into 64 groups based on sequence similarity. Analysis of the clusters revealed deep phylogenetic conservation of open reading frame organization as well as the presence of conserved sequence patterns that indicate likely utilization of the nonstandard decoding mechanisms: programmed ribosomal frameshifting (PRF) and programmed transcriptional realignment (PTR). Further enrichment of these clusters with additional homologous nucleotide sequences revealed over 6,000 candidate genes utilizing PRF or PTR. Analysis of the patterns of conservation apparently associated with nontriplet decoding revealed the presence of both previously characterized frameshift-prone sequences and a few novel ones. Since the starting point of our analysis was a set of genes with already annotated disruptions, it is highly plausible that in this study, we have identified only a fraction of all bacterial genes that utilize PRF or PTR. In addition to the identification of a large number of recoded genes, a surprising observation is that nearly half of them are expressed via PTR-a mechanism that, in contrast to PRF, has not yet received substantial attention.
Asunto(s)
Sistema de Lectura Ribosómico/genética , Genes Bacterianos/genética , Variación Genética , Sistemas de Lectura Abierta/genética , Selección Genética , Transcripción Genética/genética , Análisis por Conglomerados , Biología Computacional , Secuencia Conservada/genética , Filogenia , Proyectos PilotoRESUMEN
The IS911 bacterial transposable element uses -1 programmed translational frameshifting to generate the protein required for its mobility: translation initiated in one gene (orfA) shifts to the -1 frame and continues in a second overlapping gene (orfB), thus generating the OrfAB transposase. The A-AAA-AAG frameshift site of IS911 is flanked by two stimulatory elements, an upstream Shine-Dalgarno sequence and a downstream stem-loop. We show here that, while they can act independently, these stimulators have a synergistic effect when combined. Mutagenic analyses revealed features of the complex stem-loop that make it a low-efficiency stimulator. They also revealed the dual role of the upstream Shine-Dalgarno sequence as (i) a stimulator of frameshifting, by itself more potent than the stem-loop, and (ii) a mandatory determinant of initiation of OrfB protein synthesis on an AUU codon directly preceding the A6G motif. Both roles rely on transient base pairing of the Shine-Dalgarno sequence with the 3' end of 16S rRNA. Because of its effect on frameshifting, the Shine-Dalgarno sequence is an important determinant of the level of transposase in IS911-containing cells, and hence of the frequency of transposition.
Asunto(s)
Elementos Transponibles de ADN , Sistema de Lectura Ribosómico , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sitios de Unión , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , ARN Ribosómico 16S/metabolismo , Transposasas/biosíntesisRESUMEN
Using an approach based on the comparison of arbitrary primer polymerase chain reaction (PCR) genomic profiles from oral streptococci and Streptococcus pneumoniae strains, we identified a 434-bp genomic fragment apparently specific for S. pneumoniae. From the nucleotidic sequence of this common fragment, a pair of primers was designed and tested on a set of strains comprising the major Streptococcus species. One species, S. anginosus, gave an amplification product of the same length as S. pneumoniae. Sequence comparison of the S. anginosus and S. pneumoniae amplicons revealed several variations which were used to define a new set of primers giving a 181-bp S. pneumoniae-specific fragment. The amplified fragment contains the 5' terminal part of a gene encoding a putative sugar-specific permease and an intergenic sequence. The PCR test was evaluated on 257 strains of invasive S. pneumoniae corresponding to clinical isolates and on 153 non-pneumoniae oral streptococci strains; in addition, 3 S. pseudopneumoniae strains were tested. With these primers, an amplification product was only obtained with the S. pneumoniae strains. Moreover, the test was successfully evaluated on 10 atypical S. pneumoniae strains related to pneumococcal diseases. In this study, we therefore established the capacity of a simple PCR test to discriminate S. pneumoniae from other Streptococci (including S. pseudopneumoniae), thus allowing rapid and accurate diagnosis.
Asunto(s)
Técnicas Bacteriológicas/métodos , Infecciones Neumocócicas/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/aislamiento & purificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , ADN Bacteriano/química , ADN Bacteriano/genética , Humanos , Lactante , Persona de Mediana Edad , Datos de Secuencia Molecular , Infecciones Neumocócicas/microbiología , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Streptococcus anginosus/genética , Streptococcus pneumoniae/genética , Adulto JovenRESUMEN
We report here that YajL is associated with ribosomes and interacts with many ribosomal proteins and that a yajL mutant of Escherichia coli displays decreased translation accuracy, as well as increased dissociation of 70S ribosomes into 50S and 30S subunits after oxidative stress.
Asunto(s)
Escherichia coli/fisiología , Biosíntesis de Proteínas , Proteínas Ribosómicas/deficiencia , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Ribosomas/metabolismoRESUMEN
Nearly all members of a widespread family of bacterial transposable elements related to insertion sequence 3 (IS3), therefore called the IS3 family, very likely use programmed -1 ribosomal frameshifting to produce their transposase, a protein required for mobility. Comparative analysis of the potential frameshift signals in this family suggested that most of the insertion sequences from the IS51 group contain in their mRNA an elaborate pseudoknot that could act as a recoding stimulator. It results from a specific intramolecular interaction between an apical loop and an internal loop from two stem-loop structures. Directed mutagenesis, chemical probing, and gel mobility assays of the frameshift region of one element from the IS51 group, IS3411, provided clear evidences of the existence of the predicted structure. Modeling was used to generate a three-dimensional molecular representation of the apical loop-internal loop complex. We could demonstrate that mutations affecting the stability of the structure reduce both frameshifting and transposition, thus establishing the biological importance of this new type of RNA structure for the control of transposition level.
Asunto(s)
Escherichia coli/química , Escherichia coli/genética , Sistema de Lectura Ribosómico/genética , ARN/química , ARN/genética , Secuencia de Bases , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The translational initiation region (TIR) of the Escherichia coli rpsA gene, which encodes ribosomal protein S1, shows a number of unusual features. It extends far upstream (to position -91) of the initiator AUG, it lacks a canonical Shine-Dalgarno sequence (SD) element, and it can fold into three successive hairpins (I, II, and III) that are essential for high translational activity. Two conserved GGA trinucleotides, present in the loops of hairpins I and II, have been proposed to form a discontinuous SD. Here, we have tested this hypothesis with the "specialized ribosome" approach. Depending upon the constructs used, translation initiation was decreased three- to sevenfold upon changing the conserved GGA to CCU. However, although chemical probing showed that the mutated trinucleotides were accessible, no restoration was observed when the ribosome anti-SD was symmetrically changed from CCUCC to GGAGG. When the same change was introduced in the SD from a conventional TIR as a control, activity was stimulated. This result suggests that the GGA trinucleotides do not form a discontinuous SD. Others hypotheses that may account for their role are discussed. Curiously, we also find that, when expressed at moderate level (30 to 40% of total ribosomes), specialized ribosomes are only twofold disadvantaged over normal ribosomes for the translation of bulk cellular mRNAs. These findings suggest that, under these conditions, the SD-anti-SD interaction plays a significant but not essential role for the synthesis of bulk cellular proteins.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genes Bacterianos/genética , Proteínas Ribosómicas/genética , Secuencia de Bases , Elementos Transponibles de ADN , Proteínas de Escherichia coli/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional , Proteínas Ribosómicas/metabolismoRESUMEN
Dynamic shifts between open reading frames and the redefinition of codon meaning at specific sites, programmed by signals in mRNA, permits versatility of gene expression. Such alterations are characteristic of organisms in all domains of life and serve a variety of functional purposes. In this article, we concentrate on programmed ribosomal frameshifting, stop codon read-through and transcriptional slippage in the decoding of phage genes and bacterial mobile elements. Together with their eukaryotic counterparts, the genes encoding these elements are the richest known source of nonstandard decoding. Recent analyses revealed several novel sequences encoding programmed alterations in gene decoding and provide a glimpse of the emerging picture.
Asunto(s)
Bacteriófagos/genética , Codón/genética , Bases de Datos Factuales , Sistema de Lectura Ribosómico/genética , Secuencias Repetitivas Esparcidas/genética , Bacteriófagos/metabolismo , Mutación del Sistema de Lectura , Regulación de la Expresión GénicaRESUMEN
The discovery of programmed -1 frameshifting at the hexanucleotide shift site CGA_AAG, in addition to the classical X_XXY_YYZ heptanucleotide shift sequences, prompted a search for instances among eubacterial insertion sequence elements. IS1222 has a CGA_AAG shift site. A genetic analysis revealed that frameshifting at this site is required for transposition.
Asunto(s)
Elementos Transponibles de ADN , Sistema de Lectura Ribosómico , Rahnella/genética , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
Programmed -1 ribosomal frameshifting, involving tRNA re-pairing from an AAG codon to an AAA codon, has been reported to occur at the sequences CGA AAG and CAA AAG. In this study, using the recoding region of insertion sequence IS3, we have investigated the influence on frameshifting in Escherichia coli of the first codon of this type of motif by changing it to all other NNA codons. Two classes of NNA codons were distinguished, depending on whether they favor or limit frameshifting. Their degree of shiftiness is correlated with wobble propensity, and base 34 modification, of their decoding tRNAs. A more flexible anticodon loop very likely makes the tRNAs with extended wobble more prone to liberate the third codon base, A, for re-pairing of tRNALys in the -1 frame.
Asunto(s)
Mutación del Sistema de Lectura/genética , Biosíntesis de Proteínas , ARN de Transferencia/genética , Secuencia de Bases , Codón/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos/genética , ARN Bacteriano/genéticaRESUMEN
The IS911 bacterial transposable element has been analyzed for its mechanism of transposition and for the way it controls the expression of its genes by programmed -1 translational frameshifting. In the present study the prevalence of IS911 has been determined in the Enterobacteriaceae family and in other Gram-negative bacilli. Three variants, found in Escherichia coli clinical isolates and having mutations in the region implicated in frameshifting, were functionally characterized. All three were altered in their frameshifting and transposition abilities, suggesting that the frameshift region of IS911 may constitute a target for mutations reducing the transposition frequency of this mobile element in natural populations of E. coli.
Asunto(s)
Elementos Transponibles de ADN/genética , Escherichia coli/genética , Mutación del Sistema de Lectura , ADN Bacteriano/genética , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Regulación Bacteriana de la Expresión Génica , Humanos , Modelos Genéticos , Mutagénesis InsercionalRESUMEN
Translating ribosomes can shift reading frame at specific sites with high efficiency for gene expression purposes. The most common type of shift to the -1 frame involves a tandem realignment of two anticodons from pairing with mRNA sequence of the form X XXY YYZ to XXX YYY Z where the spaces indicate the reading frame. The predominant -1 shift site of this type in eubacteria is A AAA AAG. The present work shows that in Escherichia coli the identity of the 6 nt 3' of this sequence can be responsible for a 14-fold variation in frameshift frequency. The first 3' nucleotide has the primary effect, with, in order of decreasing efficiency, U > C > A > G. This effect is independent of other stimulators of frameshifting. It is detected with other X XXA AAG sequences, but not with several other heptameric -1 shift sites. Pairing of E. coli tRNALYS with AAG is especially weak at the third codon position. We propose that strong stacking of purines 3' of AAG stabilizes pairing of tRNALys, diminishing the chance of codon:anticodon dissociation that is a prerequisite for the realignment involved in frameshifting.