Receptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as an target given its location on the small ribosomal subunit and other properties. However, prior results suggested that RACK1 has roles both on and off the ribosome, and such an exchange might be related to its various cellular functions and hinder our ability to use RACK1 as a stable fluorescent tag for the ribosome. In addition, the kinetics of spontaneous exchange of RACK1 or any ribosomal protein from a mature ribosome in vitro remain unclear. To address these issues, we engineered fluorescently-labeled human ribosomes via RACK1, and applied bulk and single-molecule biochemical analyses to track RACK1 on and off the human ribosome. Our results demonstrate that, despite its cellular non-essentiality from yeast to humans, RACK1 readily re-associates with the ribosome, displays limited conformational dynamics, and remains stably bound to the ribosome for hours in vitro. This work sheds insight into the biochemical basis of ribosomal protein exchange on and off a mature ribosome and provides tools for single-molecule analysis of human translation.
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