RESUMEN
Recombinant protein expression is widely used to produce large quantities of protein for diverse uses including functional characterization of selected sequences and vaccination trials. In the postgenomic era, high-throughput techniques that allow us to manipulate several sequences are needed. Cloning by in vivo recombination is a technique that consists in the insertion of a linear DNA into a linearized plasmid DNA by in vivo recombination using a recA+ E. coli strain. This methodology provides high-throughput cloning with high efficiency without the need for restriction enzyme digestion. In this chapter, we describe two protocols for DNA cloning: one using in vivo recombination and the other by using restriction enzymes. We also describe the application of different conditions to produce functional proteins that needs the incorporation of the amino acid selenocysteine (Sec), like thioredoxin-glutathione reductase enzyme.
Asunto(s)
Codón/genética , Escherichia coli/genética , Proteínas Recombinantes/genética , Clonación Molecular/métodos , ADN/genética , Enzimas de Restricción del ADN/genética , Plásmidos/genética , Selenocisteína/genéticaRESUMEN
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
Selenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Sec-incorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model.
Asunto(s)
Adaptación Biológica/genética , Caenorhabditis elegans/metabolismo , Evolución Molecular , Silenciador del Gen , Redes y Vías Metabólicas/genética , Selenocisteína/metabolismo , Secuencia de Aminoácidos , Animales , Emparejamiento Base , Secuencia de Bases , Caenorhabditis elegans/genética , Codón de Terminación , Datos de Secuencia Molecular , Filogenia , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Selenocisteína/genética , Selenoproteínas/genéticaRESUMEN
In bacteria selenocysteyl-tRNA(sec) (SelC) is synthesized by selenocysteine synthase (SelA). Here we show by fluorescence anisotropy binding assays and electron microscopical symmetry analysis that the SelA-tRNA(sec) binding stoichiometry is of one tRNA(sec) molecule per SelA monomer (1:1) rather than the 1:2 value proposed previously. Negative stain transmission electron microscopy revealed a D5 pointgroup symmetry for the SelA-tRNA(sec) assembly both with and without tRNA(sec) bound. Furthermore, SelA can associate forming a supramolecular complex of stacked decamer rings, which does not occur in the presence of tRNA(sec). We discuss the structure-function relationships of these assemblies and their regulatory role in bacterial selenocysteyl-tRNA(sec) synthesis.