Structural and functional studies on drosophila ADH

The enzyme alcohol dehydrogenase of the fruit-fly Drosophila (DADH) catalyzes the same reaction as the mammalian alcohol dehydrogenases, transforming alcohols into aldehydes through the reduction of nicotinamideadenine-dinucleotide. Despite this identical chemical behaviour the enzyme's structure belongs to a different class of proteins, called short-chain dehydrogenases, which have a totally different three-dimensional architecture to the mammalian alcohol dehydrogenases. The study of the structure-fuction relationships of DADH is of interest because, while we still lack a crystal structure for the protein, large amounts of biochemicafevolutionary and genetical data have accumulated which require structural information on the enzyme for its proper interpretation.The aim of this project was two-fold: to set up a suitable system for the undertaking of protein engineering studies on DADH and to start such studies by producing and analyzing a first set of site-directed mutants. The first part of the project involved: a) creating suitable genetic vectors for the introduction of mutations, their sequencing and the expression of the mutated enzymes in yeast; b) the development of a purification method to obtain pure enzyme solutions from the expressing yeast culture; c) the development of biochemical and biophysical assays for the evaluation of the mutation's effects and, d) the construction of a three-dimensional model for the enzyme that could offer structural explanations to such effects.The second part consisted of the actual introduction of five different mutations in the enzyme's sequence, and their further evaluation using the system set up in the first place

La Traducció genètica mitocondrial i malalties associades

En humans, com en la majoria d'organismes eucariotes, la síntesi proteica té lloc simultàniament al citoplasma i en orgànuls que posseeixen un genoma propi. Els mitocondris requereixen una maquinària traduccional pròpia per sintetitzar els tretze polipèptids, codificats al genoma mitocondrial, que formen part dels complexos de la cadena respiratòria i la fosforilació oxidativa responsables de proporcionar energia a la cèl·lula. Els elements que componen aquesta maquinària es troben codificats tant al genoma mitocondrial com al nuclear i participen de manera coordinada en la traducció genètica. Mutacions en els gens que codifiquen aquests factors de l'aparell de traducció genètica mitocondrial desencadenen un ampli ventall de malalties greus en humans, caracteritzades per símptomes heterogenis que en dificulten el diagnòstic i tractament. Hi ha malalties mitocondrials humanes causades per mutacions en el DNA mitocondrial que afecten específicament els tRNA i rRNA i, a més, s'han descrit mutacions en proteïnes mitocondrials codificades en el genoma nuclear, entre les quals es troben mutacions en factors de traducció, enzims de processament i modificació dels tRNA, proteïnes mitoribosòmiques i aminoacil-tRNA-sintetases mitocondrials. La complexitat de les malalties mitocondrials, la varietat de símptomes que causen i la dificultat de manipular genèticament el DNA mitocondrial compliquen la recerca relacionada amb aquestes malalties i justifiquen la generació de models animals que permetin caracteritzar-les i desenvolupar noves estratègies terapèutiques.In humans, as in the majority of eukaryotic organisms, protein synthesis occurs simultaneously in the cytoplasm and in those organelles that possess their own genome. Mitochondria require its own translational machinery in order to synthesize the 13 polypeptides, encoded in the mitochondrial genome, which are part of the respiratory chain and the oxidative phosphorylation complexes, responsible for supplying energy to the cell. The elements that compose this machinery are encoded both in the mitochondrial and the nuclear genome, and participate in gene translation in a coordinate manner. Mutations in genes that code for these factors of the gene translation apparatus trigger a wide range of severe pathologies in humans, characterized by heterogeneous symptoms that difficult their diagnostic and treatment. There exist human mitochondrial diseases caused by mutations in the mitochondrial DNA which specifically affect tRNA and rRNA and, additionally, mutations in nuclear encoded mitochondrial proteins have been described, among which are mutations in translation factors, enzymes involved in tRNA processing and modification, mitoribosomal proteins, and aminoacyl-tRNA synthetases. The complexity of mitochondrial pathologies, the variety of symptoms they cause, and the difficulty to manipulate mitochondrial DNA complicate the research related to these diseases and justify the generation of animal models that allow their characterization and the development of new therapeutic strategies

More than an adaptor molecule: The emerging role of tRNA in cell signaling and disease

This FEBS Letters ‘FOCUS ON’ series of short reviews on tRNA captures the essence of the Barcelona BioMed Conference on Gene Translation: Fidelity and Quality Control, which was held at the Institut d’Estudis Catalans in Barcelona on December 2–4, 2013. This meeting was powered by the dramatic resurgence of interest in tRNA biochemistry following the realization that tRNA is much more than a simple adaptor of the genetic code

Aminoacyl-tRNA synthetases: a complex system beyond protein synthesis

Les aminoacil-tRNA sintetases (ARSs) són els enzims que tradueixen el codi genètic unint aminoàcids a l'RNA de transferència (ARNt) corresponent. Els tRNA aminoacilats poden ser utilitzats aleshores pel ribosoma per traduir RNA missatgers (mRNA). El rol essencial de les ARS es va establir en la dècada dels seixanta, durant l'era d'or de la biologia molecular, que va dur al descobriment del codi genètic. El paper canònic d'aquests enzims es troba actualment descrit en tots els llibres de text. Tot i això, l'interès per la funció de les ARS continua creixent extraordinàriament, a causa de les noves i inesperades funcions descobertes per a aquests enzims, per a l'ARNt i per a l'ARN en general. En aquest article descriurem els darrers progressos en l'estudi de les aminoacil-tRNA sintetases, resumirem els coneixements actuals sobre l'evolució de les ARS, introduirem els lectors en diverses facetes de la biologia cel·lular en què s'ha comprovat que les ARS tenen un paper important i discutirem les aplicacions derivades d'aquests estudis.Aminoacyl-tRNA synthetases (ARSs) are enzymes that translate the genetic code by adding amino acids to their cognate transfer RNAs (tRNA). Aminoacylated tRNAs can then be used by the ribosome to decode mRNA. The essential role of ARSs was established in the 1960s, during the golden era of molecular biology that led to the discovery of the genetic code. The canonical role of these enzymes is now described in all textbooks. Remarkably, however, interest in ARS function continues to grow as new and unexpected functions are discovered for these enzymes, for tRNA, and for RNA in general. This article describes current progress in the field of ARS research, summarizes current thinking about the evolution of ARSs, introduces the readers to the many facets of cellular biology in which ARSs play an important role, and discusses the biotechnological applications derived from these studies

Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes

The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum. We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family

A-to-I editing on tRNAs: Biochemical, biological and evolutionary implications

AbstractInosine on transfer RNAs (tRNAs) are post-transcriptionally formed by a deamination mechanism of adenosines at positions 34, 37 and 57 of certain tRNAs. Despite its ubiquitous nature, the biological role of inosine in tRNAs remains poorly understood. Recent developments in the study of nucleotide modifications are beginning to indicate that the dynamics of such modifications are used in the control of specific genetic programs. Likewise, the essentiality of inosine-modified tRNAs in genome evolution and animal biology is becoming apparent. Here we review our current understanding on the role of inosine in tRNAs, the enzymes that catalyze the modification and the evolutionary link between such enzymes and other deaminases