Studies on the in vitro synthesis of β-galactosidase: Necessary components in the ribosomal wash

Studies of the Interaction of Escherichia coli YjeQ with the Ribosome In Vitro

“…To our surprise, isolation of 70S ribosomes from E. coli and Western blotting with anti-YjeQ antibodies revealed that nearly all of the YjeQ in the cell was associated with ribosomes. Indeed, the interaction was stable to wash conditions that are typically used to remove translation factors from ribosome preparations (13). Quantitative Western blotting put the copy number of YjeQ at about 100 copies per cell, in a stoichiometry of about 1 for every 200 ribosomes, consistent with the fact that this protein has not been reported in ribosome preparations previously.…”

“…In these experiments we employed simple detergent and salt washes followed by ultracentrifugation to simply isolate ribosomes from E. coli extracts and assess the localization of YjeQ. Indeed, the interaction was stable to detergent and high salt wash conditions, conventionally used to prepare ribosomes that are substantially free from translation factors (13). Furthermore, quantitative Western blotting revealed that YjeQ was of low abundance in E. coli, possessing a protein copy number of ϳ100 copies/cell and consequently in a substoichiometric association with ribosomes (YjeQ/ribosome ratio, 1:200) (data not shown).…”

“…Crude and rapid isolation of ribosomes from E. coli cell extracts was achieved by centrifugation at 40,000 ϫ g for 1 h. The supernatant was further clarified by ultracentrifugation at 150,000 ϫ g for 2 h. Stringency washes, first with 0.5% Triton, then with 60 mM NH 4 Cl, and finally with 1 M NH 4 Cl, were performed for 2 h at 4°C, according to standard methods (13). Immunoblotting using SDS-15% polyacrylamide gels was performed as described in the legend to Fig.…”

Mechanism of protein biosynthesis in prokaryotic cells

“…2.5 times) the limiting V,,, of the process which corresponds to the 'on' rate constant of the rate-limiting step (step C in fig.1) [16]. Accordingly, the extent of this stimulation agrees well with the extent to which IF1 increases the level of fMet-tRNA binding at equilibrium [9] or the amount of protein synthesized [4]. Thus, it would appear that the kinetic stimulation observed here is sufficient, by and large, to account for the overall effect of IF1 on protein synthesis, in accordance with the widely accepted premise that initiation complex formation is the rate-limiting step in translation.…”

“…Translational initiation factor IF1 , the smallest of the three initiation factors, has been found to stimulate several partial reactions pertaining to the initiation process (reviews, see [l-3]) and is required for optimal in vitro protein synthesis [4]. However, no autonomous role has been found for IFl, whose overall mechanism of action remains rather elusive.…”