Reconstitution of the human SRP system and quantitative and systematic analysis of its ribosome interactions.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
08 04 2019
08 04 2019
Historique:
accepted:
02
01
2019
revised:
20
12
2018
received:
10
10
2018
pubmed:
17
1
2019
medline:
12
10
2019
entrez:
17
1
2019
Statut:
ppublish
Résumé
Co-translational protein targeting to membranes depends on the regulated interaction of two ribonucleoprotein particles (RNPs): the ribosome and the signal recognition particle (SRP). Human SRP is composed of an SRP RNA and six proteins with the SRP GTPase SRP54 forming the targeting complex with the heterodimeric SRP receptor (SRαβ) at the endoplasmic reticulum membrane. While detailed structural and functional data are available especially for the bacterial homologs, the analysis of human SRP was impeded by the unavailability of recombinant SRP. Here, we describe the large-scale production of all human SRP components and the reconstitution of homogeneous SRP and SR complexes. Binding to human ribosomes is determined by microscale thermophoresis for individual components, assembly intermediates and entire SRP, and binding affinities are correlated with structural information available for all ribosomal contacts. We show that SRP RNA does not bind to the ribosome, while SRP binds with nanomolar affinity involving a two-step mechanism of the key-player SRP54. Ultrasensitive binding of SRP68/72 indicates avidity by multiple binding sites that are dominated by the C-terminus of SRP72. Our data extend the experimental basis to understand the mechanistic principles of co-translational targeting in mammals and may guide analyses of complex RNP-RNP interactions in general.
Identifiants
pubmed: 30649417
pii: 5289490
doi: 10.1093/nar/gky1324
pmc: PMC6451106
doi:
Substances chimiques
Receptors, Cytoplasmic and Nuclear
0
Receptors, Peptide
0
SRP54 protein, human
0
SRP68 protein, human
0
SRP72 protein, human
0
Signal Recognition Particle
0
signal peptide receptor
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
3184-3196Informations de copyright
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
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