Cargando…

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,...

Descripción completa

Detalles Bibliográficos
Autores principales: Johnson, Alex G., Lapointe, Christopher P., Wang, Jinfan, Corsepius, Nicholas C., Choi, Junhong, Fuchs, Gabriele, Puglisi, Joseph D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6573788/
https://www.ncbi.nlm.nih.gov/pubmed/31023766
http://dx.doi.org/10.1261/rna.071217.119
_version_ 1783427785066283008
author Johnson, Alex G.
Lapointe, Christopher P.
Wang, Jinfan
Corsepius, Nicholas C.
Choi, Junhong
Fuchs, Gabriele
Puglisi, Joseph D.
author_facet Johnson, Alex G.
Lapointe, Christopher P.
Wang, Jinfan
Corsepius, Nicholas C.
Choi, Junhong
Fuchs, Gabriele
Puglisi, Joseph D.
author_sort Johnson, Alex G.
collection PubMed
description 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.
format Online
Article
Text
id pubmed-6573788
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher Cold Spring Harbor Laboratory Press
record_format MEDLINE/PubMed
spelling pubmed-65737882020-07-01 RACK1 on and off the ribosome Johnson, Alex G. Lapointe, Christopher P. Wang, Jinfan Corsepius, Nicholas C. Choi, Junhong Fuchs, Gabriele Puglisi, Joseph D. RNA Article 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. Cold Spring Harbor Laboratory Press 2019-07 /pmc/articles/PMC6573788/ /pubmed/31023766 http://dx.doi.org/10.1261/rna.071217.119 Text en © 2019 Johnson et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Article
Johnson, Alex G.
Lapointe, Christopher P.
Wang, Jinfan
Corsepius, Nicholas C.
Choi, Junhong
Fuchs, Gabriele
Puglisi, Joseph D.
RACK1 on and off the ribosome
title RACK1 on and off the ribosome
title_full RACK1 on and off the ribosome
title_fullStr RACK1 on and off the ribosome
title_full_unstemmed RACK1 on and off the ribosome
title_short RACK1 on and off the ribosome
title_sort rack1 on and off the ribosome
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6573788/
https://www.ncbi.nlm.nih.gov/pubmed/31023766
http://dx.doi.org/10.1261/rna.071217.119
work_keys_str_mv AT johnsonalexg rack1onandofftheribosome
AT lapointechristopherp rack1onandofftheribosome
AT wangjinfan rack1onandofftheribosome
AT corsepiusnicholasc rack1onandofftheribosome
AT choijunhong rack1onandofftheribosome
AT fuchsgabriele rack1onandofftheribosome
AT puglisijosephd rack1onandofftheribosome