RESUMEN
tRNA synthetases are responsible for decoding the molecular information, from codons to amino acids. Seryl-tRNA synthetase (SerRS), besides the five isoacceptors of tRNASer, recognizes tRNA[Ser]Sec for the incorporation of selenocysteine (Sec, U) into selenoproteins. The selenocysteine synthesis pathway is known and is dependent on several protein-protein and protein-RNA interactions. Those interactions are not fully described, in particular, involving tRNA[Ser]Sec and SerRS. Here we describe the molecular interactions between the Escherichia coli Seryl-tRNA synthetase (EcSerRS) and tRNA[Ser]Sec in order to determine their specificity, selectivity and binding order, leading to tRNA aminoacylation. The dissociation constant of EcSerRS and tRNA[Ser]Sec was determined as (126 ± 20) nM. We also demonstrate that EcSerRS binds initially to tRNA[Ser]Sec in the presence of ATP for further recognition by E. coli selenocysteine synthetase (EcSelA) for Ser to Sec conversion. The proposed studies clarify the mechanism of tRNA[Ser]Sec incorporation in Bacteria as well as of other domains of life.
Asunto(s)
Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN de Transferencia Aminoácido-Específico/metabolismo , ARN de Transferencia de Cisteína/metabolismo , Serina-ARNt Ligasa/metabolismo , Transferasas/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Escherichia coli/genética , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia de Cisteína/genética , Serina-ARNt Ligasa/genética , Termodinámica , Aminoacilación de ARN de Transferencia/genética , Transferasas/genéticaRESUMEN
In Bacillus subtilis, the tRNACys lacks an encoded CCA 3' end. To gain insight into the role of CCAase and RNases in tRNACys processing, several mutant strains were generated. Northern blot and RT-PCR results suggest that enzymes other than CCAase can participate in CCA addition at the 3' end of the immature tRNACys.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , ARN Nucleotidiltransferasas/deficiencia , ARN Bacteriano/genética , ARN de Transferencia de Cisteína/genética , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Conformación de Ácido Nucleico , ARN Nucleotidiltransferasas/genética , Procesamiento Postranscripcional del ARN , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN de Transferencia de Cisteína/química , ARN de Transferencia de Cisteína/metabolismoRESUMEN
American foulbrood (AFB) caused by Paenibacillus larvae is the most destructive honeybee bacterial disease and its dissemination via commercial bee pollen is an important mechanism for the spread of this bacterium. Because Mexico imports bee pollen from several countries, we developed a tRNACys-PCR strategy and complemented that strategy with MALDI-TOF MS and amplicon-16S rRNA gene analysis to evaluate the presence of P. larvae in pollen samples. P. larvae was not detected when the tRNACys-PCR approach was applied to spore-forming bacterial colonies obtained from three different locations and this result was validated by bacterial identification via MALDI-TOF MS. The genera identified in the latter analysis were Bacillus (fourteen species) and Paenibacillus (six) species. However, amplicon-16S rRNA gene analysis for taxonomic composition revealed a low presence of Paenibacillaceae with 0.3 to 16.2% of relative abundance in the commercial pollen samples analyzed. Within this family, P. larvae accounted for 0.01% of the bacterial species present in one sample. Our results indicate that the tRNACys-PCR, combined with other molecular tools, will be a useful approach for identifying P. larvae in pollen samples and will assist in controlling the spread of the pathogen.
Asunto(s)
Abejas/microbiología , Paenibacillus larvae/genética , Polen/microbiología , ARN Bacteriano/genética , ARN de Transferencia de Cisteína/genética , Animales , Bacillus/genética , Técnicas de Amplificación de Ácido Nucleico , Paenibacillus larvae/aislamiento & purificación , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estados UnidosRESUMEN
Selenium (Se) is an essential trace element for several organisms and is mostly present in proteins as L-selenocysteine (Sec or U). Sec is synthesized on its L-seryl-tRNASec to produce Sec-tRNASec molecules by a dedicated selenocysteine synthesis machinery and incorporated into selenoproteins at specified in-frame UGA codons. UGA-Sec insertion is signaled by an mRNA stem-loop structure called the SElenoCysteine Insertion Sequence (SECIS). tRNASec transcription regulation and folding have been described showing its importance to Sec biosynthesis. Here, we discuss structural aspects of Sec-tRNASec and its role in Sec biosynthesis as well as Sec incorporation into selenoproteins. Defects in the Sec biosynthesis or incorporation pathway have been correlated with pathological conditions.
Asunto(s)
ARN de Transferencia de Cisteína/genética , Selenocisteína/biosíntesis , Animales , Codón de Terminación/química , Codón de Terminación/genética , Codón de Terminación/metabolismo , Humanos , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia de Cisteína/química , ARN de Transferencia de Cisteína/metabolismo , Selenocisteína/genéticaRESUMEN
We generated a conditional CCase mutant of Bacillus subtilis to explore the participation in vivo of the tRNA nucleotidyltransferase (CCA transferase or CCase) in the maturation of the single-copy tRNA(Cys), which lacks an encoded CCA 3' end. We observed that shorter tRNA(Cys) species, presumably lacking CCA, only accumulated when the inducible Pspac : cca was introduced into an rnr mutant strain, but not in combination with pnp. We sequenced the tRNA 3' ends produced in the various mutant tRNA(Cys) species to detect maturation and decay intermediates and observed that decay of the tRNA(Cys) occurs through the addition of poly(A) or heteropolymeric tails. A few clones corresponding to full-size tRNAs contained either CCA or other C and/or A sequences, suggesting that these are substrates for repair and/or decay. We also observed editing of tRNA(Cys) at position 21, which seems to occur preferentially in mature tRNAs. Altogether, our results provide in vivo evidence for the participation of the B. subtilis cca gene product in the maturation of tRNAs lacking CCA. We also suggest that RNase R exoRNase in B. subtilis participates in the quality control of tRNA.