The present study focuses on the initiation phase of protein synthesis and the generation of aptamers as candidates to reduce the physiological function of the isolated N terminal domain (NTD) of initiation factor 3 (IF-3). Through molecular biology techniques, the correct cloning of the gene coding for IF-3 NTD in plasmid pET24a was verified. This plasmid was used to transform the bacterial model organism Escherichia coli BL21 and thus the massive production of the IF-3 NTD was assessed. Using affinity chromatography techniques, the NTD of the IF-3 was isolated, obtaining high purity degrees and production yields. The purified NTD was used to generate aptamers with the SELEX technique (Systematic Evolution of Ligands by Exponential Enrichment). Five molecules with binding potential to the NTD of IF-3 were found. The present investigation provides the bases to study the interaction of the NTD of IF-3 with the aptamers in cell-free system and thus to evaluate their inhibitory potential. It is expected that these molecules behave as potential new drugs, and therefore they might contribute to cope for the need of new antibiotics. / Tesis

Las proteínas ribosomales S13 y L33 pertenecen respectivamente a las subunidades 30S y 50S del ribosoma bacteriano. S13 se posiciona en cercanía a los factores de iniciación (IF1 e IF3). L33 se encuentra opuesta a S13 en la subunidad mayor. El presente estudio, busca evaluar las proteínas S13 y L33 marcadas fluorescentemente durante la iniciación de la síntesis proteica, para generar un sistema experimental que permita analizar cambios estructurales en las subunidades ribosomales, especialmente durante la iniciación de la traducción y en función de la unión de antibióticos. Mediante técnicas recombinantes y cromatografía de intercambio iónico, se expresaron y purificaron ambas proteínas ribosomales. Se obtuvieron rendimientos de la producción de proteína L33 en el rango de miligramos por g de Escherichia coli, e índices de pureza sobre el 98%. S13 fue producido de manera similar y se obtuvieron proteínas puras en el rango de miligramos por g de Escherichia coli con una pureza de 99%. Ambas proteínas ribosomales fueron modificadas con compuestos fluorescentes compatibles con mediciones de FRET (De sus siglas en inglés, Föester Resonance Energy Transfer). En condiciones controladas S13 logró insertarse en la subunidad 30S, mientras que experimentos de sedimentación indicaron que L33 fluorescente no se unía a la subunidad 50S. Se realizaron mediciones FRET entre la subunidad 30S modificada con S13 fluorescente y los factores de iniciación IF1 e IF3. La medición FRET entre S13(Atto-540Q) e IF3-CTD(Atto-488) se utilizó para medir la interacción de estreptomicina, espectinomicina y GE81112, antibióticos que se unen a la subunidad menor del ribosoma. Estreptomicina se une rápidamente a la subunidad 30S y ocasiona un acercamiento de S13(Atto-540Q) al IF3-CTD(Atto-488) mientras que GE81112, y en menor medida espectinomicina, promueven un alejamiento de la proteína ribosomal del factor de iniciación. Ambos movimientos son compatibles con rotaciones de la cabeza de la subunidad 30S, previamente descritos como fundamentales para el funcionamiento del ribosoma. El presente estudio de investigación brinda un sistema experimental novedoso para estudiar cambios estructurales en la subunidad menor en función de la unión de antibióticos al ribosoma. / The ribosomal proteins S13 and L33 belong respectively to the 30S and 50S subunits of the bacterial ribosome. S13 is positioned close to the initiation factors (IF1 and IF3). L33 is opposite and near to S13 in the major subunit. The present study, the aim is to evaluate the fluorescently labeled S13 and L33 proteins during the initiation of protein synthesis, aiming to build an experimental system that allows the analysis of structural changes in the ribosomal subunits, especially during the initiation of translation and with the binding of antibiotics. Using recombinant techniques and ion exchange chromatography, both ribosomal proteins were expressed and purified. Production yields of pure proteins of L33 were obtained in the range of milligrams per g of Escherichia coli, in addition, of 98% purity indices. S13 was produced in a similar manner, obtaining milligrams of pure protein per g of Escherichia coli with a purity of 99%. Both ribosomal proteins were modified with fluorescent compounds compatible with measurements of FRET (Föester Resonance Energy Transfer). Under controlled conditions S13 was able to insert into the 30S subunit, while sedimentation experiments indicated that fluorescent L33 did not bind to the 50S subunit. The 30S subunit modified with fluorescent S13 showed various FRET signals in combination with the initiation factors IF1 and IF3. These signals were used to measure the interaction of streptomycin, spectinomycin and GE81112, antibiotics that bind to the minor subunit of the ribosome. Streptomycin binds rapidly to the 30S subunit and causes an approximation of S13(Atto-540Q) to IF3-CTD(Atto-488) while GE81112, and to a lesser extent spectinomycin, promote a distancing of the ribosomal protein from the initiation factor. Both movements can account for rotations of the head of the 30S subunit, previously described as fundamental for the functioning of the ribosome. The present research provides a novel approach to study structural changes in the minor subunit according to the binding of antibiotics to the ribosome.