The stoichiometry of Smp B within both complexes was evaluated by Western blot.
Band intensity analysis indicates that the amount of Smp B present in the preaccommodated state is almost twice that recovered after accommodation (2 vs. 1B), suggesting that accommodation triggers the departure of one ribosome-bound protein. Images of frozen-hydrated ribosomal complexes were recorded using a La B6 microscope operating at 200 k V.
Also, the movement of the aminoacyl-end of tm RNA into the A-site upon accommodation of the TLD is expected to produce a steric clash of the molecule of Smp B bound to the 50S with the P-site t RNA (Gutmann et al. Therefore, we can predict that the molecule of Smp B bound to the 50S subunit has to move away, but the questions are when and how it happens.
To address these questions, we used cryo-EM three-dimesional reconstruction to study a ribonucleoprotein complex made of the TLD as well as the extended helix H2 of tm RNA bound to Smp B into stalled ribosomes.
Therefore, in order to reduce its flexibility and increase its occupancy in the stalled ribosomes, we decided to work with a truncated version of tm RNA, depleted from its four pseudoknots and internal coding portion. After purification by size exclusion chromatography, ribosome complexes were analyzed by SDS-PAGE.
Recent biochemical and structural data indicate that Smp B provides contacts in place of the t RNA anti-codon stem, being a functional mimic of the pairing between a codon and an anti-codon at the ribosome decoding center (Nonin-Lecomte et al. At an early stage during -translation, two molecules of Smp B are recovered per stalled ribosome, one on each subunit and both contacting the “t RNA-like domain” (TLD) of tm RNA (Hallier et al. The truncated m RNA is removed and degraded while the stalled ribosome switches the translation process to the tm RNA coding sequence resume codon, adding the tag peptide to the C terminus of the nascent polypeptide. 1997) combined with cryo-electron microscopy (cryo-EM) reconstructions of the RNA backbone within the electron density of a preaccommodated complex (Kaur et al. During canonical translation, initial selection corresponds to the reversible association of the t RNA anti-codon with the codon in the 30S A-site, while the aminoacyl-acceptor end is still bound to elongation factor Tu (EF-Tu) (Ogle and Ramakrishnan 2005). After the release of the inorganic phosphate, EF-Tu rearranges into a GDP-bound conformation that dissociates from the aminoacyl end of the t RNA and from the ribosome.
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