Predicción bioinformática y validación experimental de genes RNA regulatorios pequeños en la bactería extremófila Acidithiobacillus ferrooxidans
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Fecha
2010
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Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
RESUMEN: En bacterias los RNA reguladores pequeños (srRNA – “small regulatory RNA”) controlan la expresión génica, usualmente a nivel post-transcripcional. Esto lo hacen actuando como RNAs anti-sentido uniéndose al transcrito (mRNA) de los genes blanco (mRNAs) o interactuando con las proteínas reguladoras. Los srRNAs están involucrados en la regulación de una amplia variedad de procesos, tales como la replicación de plasmidios, el transporte, la replicación viral, la virulencia bacteriana y algunos de los circuitos genéticos globales que responden a cambios ambientales. Desde su descubrimiento, hace unos pocos años, se ha comprobado que se encuentran presentes en una variedad de organismos distintos. Sin embargo, su detección/predicción utilizando herramientas bioinformática es particularmente desafiante dado que no codifican para proteínas, son poco conservados a nivel nucleotídico y sólo algunos exhiben estructura secundaria conservada.
En este trabajo utilizamos herramientas computacionales para predecir los srRNAs en la secuencia genómica de la bacteria extremófila Acidithiobacillus ferrooxidans, uno de los microorganismos más utilizados en la recuperación biológica de metales a partir de minerales azufrados, y herramientas de biología molecular para validarlos. La estrategia empleada involucra en primer lugar un análisis de genómica comparativa utilizando los genomas de Acidithiobacillus caldus y Acidithiobacillus thiooxidans (secuenciados por Fundación Ciencia para la Vida [88]) para identificar candidatos de srRNA conservados en las regiones intergénicas en bacterias cercanas filogenéticamente. Las regiones intergénicas conservadas fueron extraídas del genoma de A. ferrooxidans e investigadas para determinar cuáles de las secuencias son complementarias a genes que codifican proteínas y se asocian a promotores sigma 70 predichos. En segundo término, se estudió la expresión de los candidatos srRNAs por Northern blot y “RACE”. Como producto de este análisis se derivaron 11 nuevos srRNA en A. ferrooxidans.
Adicionalmente, se ha desarrollado un nuevo programa para identificar posibles RNAs blancos de srRNA. Este programa, llamado “Kissing Complex”, se enfoca en la
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búsqueda de conexiones anti-sentido entre srRNAs y mRNAs (RNAs blancos) en la región simple hebra, específicamente, donde el RNA forma lazos o “loops”.
La identificación de los srRNAs potenciales entrega información relevante y novedosa en relación a la regulación de la expresión génica en A. ferrooxidans, permitiendo ampliar las áreas de investigación abordadas hasta la fecha y abriendo nuevas ventanas en los estudios biológicos de este microorganismo, contribuyendo así a nuestra comprensión del inusual metabolismo de las bacterias acidófilas y, eventualmente, a nuestro entendimiento de la biolixiviación.
ABSTRACT: All bacteria contain small regulatory RNAs (srRNAs), ranging in size from 50 to 500 nucleotides, which control gene expression. srRNAs are proving to be multifunctional and have provided explanations for a number of previously mysterious regulatory effects. Phages and plasmids have long been recognized to use antisense RNA regulators but now sRNAs are being discovered in all bacterial genomes, including pathogens. In eukaryotic cells, microRNAs and RNAi parallel in many ways the bacterial srRNAs, confirming that this level of regulation is widespread. However, the computational discovery of srRNAs is particularly challenging since they do not encode protein products, are poorly conserved at the sequence level and only some exhibit conserved secondary structure. This thesis describes computational predictions and experimental validations of srRNAs in the chemolithoautotrophic acidophilic genera of bacteria termed "Acidithiobacilli". Acidithiobacilli are known to play an important role in the industrial process of metal recovery termed "bioleaching" or "biomining". I characterized the sequence and genomic features of these RNAs and incorporated the identified characteristics in a predictive scheme by employing the following principles: 1) I focused on intergenic regions, defined between annotated genes based on the A. ferrooxidans database. (2) Within these regions, I searched for transcription initiation and termination signals that are widely used by the E. coli transcription machinery and that were also observed in the known srRNAs genes. We focused on promoter DNA sequences recognized by the major RNA polymerase sigma factor, σ70, and on Rho-independent terminators, in which the termination signal resides in specific sequence and structural features of the RNA. (3) Among the predicted sequences, I chose those in which the distance between the predicted promoter and terminator was 50–500 base pairs. (4) The predicted sequences obtained were compared to genome sequences of other “Acidithiobacilli”, and those that showed significant conservation XII were selected. This screen resulted in the identification of 30 candidate srRNAs encoding genes. Experimental validation is presented for 11 novel srRNAs. Additionally, I developed a new computational program to identify potential target genes for predicted srRNA. This program, called "Kissing Complex", finds antisense connections between srRNAs and mRNA (target RNAs) in the single strand regions of both molecules, specifically, where the RNA forms loops. SrRNAs are known to control gene expression in a wide variety of microorganisms, usually at the post-transcriptional level, by acting as antisense RNAs that bind targeted mRNAs or by interacting with regulatory proteins. SrRNAs are involved in the regulation of a large variety of processes such as plasmid replication, transposition and global genetic circuits that respond to environmental changes. It is expected that future work aimed at the elucidation of the function of the srRNAs described herein will contribute to our understanding of the unusual metabolism of acidophilic bacteria and ultimately to our knowledge of bioleaching.
ABSTRACT: All bacteria contain small regulatory RNAs (srRNAs), ranging in size from 50 to 500 nucleotides, which control gene expression. srRNAs are proving to be multifunctional and have provided explanations for a number of previously mysterious regulatory effects. Phages and plasmids have long been recognized to use antisense RNA regulators but now sRNAs are being discovered in all bacterial genomes, including pathogens. In eukaryotic cells, microRNAs and RNAi parallel in many ways the bacterial srRNAs, confirming that this level of regulation is widespread. However, the computational discovery of srRNAs is particularly challenging since they do not encode protein products, are poorly conserved at the sequence level and only some exhibit conserved secondary structure. This thesis describes computational predictions and experimental validations of srRNAs in the chemolithoautotrophic acidophilic genera of bacteria termed "Acidithiobacilli". Acidithiobacilli are known to play an important role in the industrial process of metal recovery termed "bioleaching" or "biomining". I characterized the sequence and genomic features of these RNAs and incorporated the identified characteristics in a predictive scheme by employing the following principles: 1) I focused on intergenic regions, defined between annotated genes based on the A. ferrooxidans database. (2) Within these regions, I searched for transcription initiation and termination signals that are widely used by the E. coli transcription machinery and that were also observed in the known srRNAs genes. We focused on promoter DNA sequences recognized by the major RNA polymerase sigma factor, σ70, and on Rho-independent terminators, in which the termination signal resides in specific sequence and structural features of the RNA. (3) Among the predicted sequences, I chose those in which the distance between the predicted promoter and terminator was 50–500 base pairs. (4) The predicted sequences obtained were compared to genome sequences of other “Acidithiobacilli”, and those that showed significant conservation XII were selected. This screen resulted in the identification of 30 candidate srRNAs encoding genes. Experimental validation is presented for 11 novel srRNAs. Additionally, I developed a new computational program to identify potential target genes for predicted srRNA. This program, called "Kissing Complex", finds antisense connections between srRNAs and mRNA (target RNAs) in the single strand regions of both molecules, specifically, where the RNA forms loops. SrRNAs are known to control gene expression in a wide variety of microorganisms, usually at the post-transcriptional level, by acting as antisense RNAs that bind targeted mRNAs or by interacting with regulatory proteins. SrRNAs are involved in the regulation of a large variety of processes such as plasmid replication, transposition and global genetic circuits that respond to environmental changes. It is expected that future work aimed at the elucidation of the function of the srRNAs described herein will contribute to our understanding of the unusual metabolism of acidophilic bacteria and ultimately to our knowledge of bioleaching.
Notas
Tesis (Doctor en Biotecnología)
Palabras clave
ARN., Bacterias -- Metabolismo.