Characterization of RNA alterations in Escherichia coli under oxidative stress and their effects over translation of the genetic message
In recent years, different researchers have proposed that translational regulation is central to the response to some stress conditions, during either its initiation and/or elongation steps. However, there is controversy over some of proposed mechanisms. One of the important hostile conditions confr...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2020 |
| País: | Chile |
| OAI Identifier: | oai:repositorio.anid.cl:10533/246361 |
| Acceso en línea: | https://hdl.handle.net/10533/246361 |
| Access Level: | acceso abierto |
| Palabra clave: | Ciencias Naturales Otras Ciencias Naturales |
| Sumario: | In recent years, different researchers have proposed that translational regulation is central to the response to some stress conditions, during either its initiation and/or elongation steps. However, there is controversy over some of proposed mechanisms. One of the important hostile conditions confronted by bacteria is oxidative stress. Bacteria are often exposed to various environments and anthropogenic factors that favor the production of oxidative molecules, which in some conditions can produce oxidative stress. Consequently, these microorganisms have developed mechanisms to overcome this stress at diverse environments, such as inside of macrophages from humans or other animals. For many years, the study of these mechanisms has focused on transcriptional changes triggered by the stress. However, the RNA components involved in translation are not static and also respond to changes in the cellular environment. In fact, there are examples of alterations induced by oxidative stress conditions to the main three types of RNA key to translation: mRNA, tRNA, and rRNA. This work tries to contribute to that discussion, determining changes in the components of the Escherichia coli translation machinery under oxidative stress, focusing our analysis on RNA molecules and how these changes affect the translation of the genetic message. Our analyzes show that oxidative stress inhibit initiation of translation from canonical mRNA and, at the same time, activates translation initiation from leaderless mRNA (lmRNA). Canonical translation seems to be inhibited by the accumulation of (p) ppGpp. Contrary to what other have been proposed, translation of lmRNA does not depend on MazF endoribonuclease, which may, nevertheless, promote the generation of some of the lmRNA through digestion of canonical mRNA. As an alternative mechanism, we propose that translation of lmRNA may be activated by the production of lighter ribosomal particles which we have observed on sedimentation profiles. Finally, we used a library of GFP variants to estimate the efficiency of translation for each codon during elongation. This allows us to study protein synthesis over time, comparing the efficiency of translation of all codons between minimal media and oxidative stress conditions. From this analysis we observe that under oxidative stress, some codons (TCC, GGA, CTC, CCT and CCC) maintain or increase their efficiency of translation, despite the general inhibition of the canonical mRNAs mentioned above. Based on these and other results we propose that inhibition of canonical translation initiation is stronger than inhibition of elongation. |
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