Reconstitution of the human SRP system and quantitative and systematic analysis of its ribosome interactions

Wild, Klemens; Juaire, Keven D.; Soni, Komal; Shanmuganathan, Vivekanandan; Hendricks, Astrid; Segnitz, Bernd; Beckmann, Roland und Sinning, Irmgard (2019): Reconstitution of the human SRP system and quantitative and systematic analysis of its ribosome interactions. In: Nucleic Acids Research, Bd. 47, Nr. 6: S. 3184-3196

Abstract

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 (SR) 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.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Medizin
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• ISSN: 0305-1048
• Sprache: Englisch