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

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 (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.

The DEAD-box RNA helicase Ded1 from yeast is associated with the signal recognition particle (SRP), and its enzymatic activity is regulated by SRP21

“…Moreover, there was variability in the stimulatory effects with different RNA preparations, which probably reflected variability in the folding of the RNAs during preparation. Indeed, others have shown that the smaller human 7SL RNA is difficult to recover as a homogeneous structure in vitro (41). Thus, to facilitate comparisons we normalized the activity relative to that of Ded1 with the RNA alone and used the same RNA preparations for comparisons.…”

“…In yeast and metazoans, SRP14 and the Alu domain of the SRP RNA play an important role in this “pausing” by blocking the GTP-dependent elongation factor eEF2 from binding at the GTPase-associated center near the mRNA entry channel at the interface between the 40S and 60S ribosomes (37,39,40). The interactions with the ribosomes depend on the SRP proteins; human 7SL RNA does not bind the ribosomes by itself (41). The SRP-ribosome complex eventually associates with the Sec61 translocon on the ER membrane, the SRP dissociates from the ribosome and translation continues with the polypeptide inserted into the ER lumen or membrane.…”

A ribosome-associated chaperone enables substrate triage in a cotranslational protein targeting complex

“…The molecular mechanism underlying this selectivity is poorly understood. While earlier models suggested that SRP binds weakly to RNCs without a strong signal sequence, mammalian SRP was found to bind tightly to RNCs with or without a signal sequence, with equilibrium dissociation constants (K d ) of <10 nM 14,15 . These observations suggested that even signalless ribosomes could be bound by SRP at its in vivo concentration (~500 nM) 16 .…”

MetAP-like Ebp1 occupies the human ribosomal tunnel exit and recruits flexible rRNA expansion segments

“…Human non-translating 80S ribosomes were isolated from HeLa cells in a protocol as described previously 43 that we adapted from a large-scale setup 44 . Briefly, HeLa cells were grown in suspension cultures and harvested cells (1 × 10 8 cells per 100 mL) were lysed with detergent.…”