No abstract

During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1–RpL5-N–RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1–RpL5–RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP.

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The eukaryotic signal recognition particle (SRP) and its receptor (SR) play a central role in co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. The SR is a heterodimeric complex assembled by the two GTPases SR␣ and SR␤, which is membrane-anchored. Here we present the 2.45-Å structure of mammalian SR␤ in its Mg 2؉ GTP-bound state in complex with the minimal binding domain of SR␣ termed SRX. SR␤ is a member of the Ras-GTPase superfamily closely related to Arf and Sar1, while SRX belongs to the SNARE-like superfamily with a fold also known as longin domain. SRX binds to the P loop and the switch regions of SR␤-GTP. The binding mode and structural similarity with other GTPase-effector complexes suggests a co-GAP (GTPase-activating protein) function for SRX. Comparison with the homologous yeast structure and other longin domains reveals a conserved adjustable hydrophobic surface within SRX which is of central importance for the SR␤-GTP:SRX interface. A helix swap in SRX results in the formation of a dimer in the crystal structure. Based on structural conservation we present the SR␤-GTP:SRX structure as a prototype for conserved interactions in a variety of GTPase regulated targeting events occurring at endomembranes. Nascent chains of secretory and membrane proteins are targeted via the ribonucleoprotein particle SRP 2 and the interaction with its receptor (SR) to the translocation machinery within the endoplasmic reticulum (ER) membrane (1, 2). The function of the SRP system is described by the SRP cycle (3, 4). SRP recognizes N-terminal hydrophobic signal sequences as soon as they emerge from the ribosomal polypeptide exit tunnel. The complex is targeted to the SR at the membrane, and the ribosome-nascent chain (RNC) complex is transferred to the translocon. Upon GTP hydrolysis in SRP and SR the complex dissociates.The eukaryotic SR consists of the two GTPases SR␣ and SR␤ (1, 5). SR␣ is a multidomain SRP GTPase with a characteristic low affinity for nucleotide (ϳ10 M) and is stable in the nucleotide-free form (6 -8). The C-terminal part of SR␣ (FtsY in archaea and eubacteria) contains the stable NG domain, which binds to the respective NG domain of SRP (SRP54, Ffh in eubacteria). Several structures of the isolated NG domains revealed the basis for the SRP GTPase cycle (for reviews, see Refs. 2-4 and 9), and the complex of the two NG domains shows a remarkably symmetric heterodimer with the nucleotides in direct contact at the center of the interface (10, 11). The N-terminal part of SR␣ is responsible for tethering SR␣ to the ER membrane bound SR␤ (12). The interaction localizes to the globular SRX domain of SR␣ comprising the first 130 residues (13). SRX has been described as effector for SR␤ and only binds to the GTP-bound form of the GTPase (13,14). The SRX domain belongs to the SNARE-like superfamily including the N-terminal domains of non-syntaxin SNAREs, also known as longin domains (13, 15). Longin domains have been proposed to regulate a variety of membrane trafficki...

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.