• S13 y L33 como proteínas sensoras de movimientos de las subunidades ribosomales durante la iniciación de la síntesis proteica en bacterias

      Milon Mayer, Pohl Luis; Cuestas Quiroz, Flavia Alejandra; Sánchez Ato, Luis Andrés; Universidad Peruana de Ciencias Aplicadas (UPC) (Universidad Peruana de Ciencias Aplicadas (UPC), 2018-02-01)
      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.
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