Abstract
Cell-free protein synthesis, which mimics the biological protein
production system, allows rapid expression of proteins without the need
to maintain a viable cell. Nevertheless, cell free protein expression
relies on active in vivo translation machinery including ribosomes and
translation factors. Here, we examined the integrity of the protein
synthesis machinery, namely the functionality of ribosomes, during (i)
the cell-free extract preparation and (ii) the performance of in vitro
protein synthesis by analyzing crucial components involved in
translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and
ribosomal protein Si, we show that processing of a cell-free extract
results in no substantial alteration of the translation machinery.
Moreover, we reveal that the 16S rRNA is specifically cleaved at helix
44 during in vitro translation reactions, resulting in the removal of
the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate
in the cell-free system. We demonstrate that the specific cleavage of
the 16S rRNA is triggered by the decreased concentrations of Mg2+. In
addition, we provide evidence that helix 44 of the 30S ribosomal subunit
serves as a point-of-entry for ribosome degradation in Escherichia coli.
Our results suggest that Mg2+ homeostasis is fundamental to preserving
functional ribosomes in cell-free protein synthesis systems, which is of
major importance for cell-free protein synthesis at preparative scale,
in order to create highly efficient technical in vitro systems.
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