RACK1 on and off the ribosome

Receptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein (RP) implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as a target given its location on the small ribosomal subunit and other properties. However,...

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Bibliographic Details
Published in:RNA (Cambridge) 2019-07, Vol.25 (7), p.881-895
Main Authors: Johnson, Alex G, Lapointe, Christopher P, Wang, Jinfan, Corsepius, Nicholas C, Choi, Junhong, Fuchs, Gabriele, Puglisi, Joseph D
Format: Article
Language:English
Subjects:
HeLa Cells
Humans
Neoplasm Proteins - chemistry
Neoplasm Proteins - genetics
Neoplasm Proteins - metabolism
Protein Binding
Protein Biosynthesis
Receptors for Activated C Kinase - chemistry
Receptors for Activated C Kinase - genetics
Receptors for Activated C Kinase - metabolism
Ribosomal Proteins - genetics
Ribosomal Proteins - metabolism
Ribosomes
Ribosomes - metabolism
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
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Summary:Receptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein (RP) implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as a 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 RP 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 nonessentiality from yeast to humans, RACK1 readily reassociates 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 RPs exchange on and off a mature ribosome and provides tools for single-molecule analysis of human translation.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.071217.119