Interaction of signal-recognition particle 54 GTPase domain and signal-recognition particle RNA in the free signal-recognition particle

Abstract

The signal-recognition particle (SRP) is a ubiquitous protein-RNA complex that targets proteins to cellular membranes for insertion or secretion. A key player in SRP-mediated protein targeting is the evolutionarily conserved core consisting of the SRP RNA and the multidomain protein SRP54. Communication between the SRP54 domains is critical for SRP function, where signal sequence binding at the M domain directs receptor binding at the GTPase domain (NG domain). These SRP activities are linked to domain rearrangements, for which the role of SRP RNA is not clear. In free SRP, a direct interaction of the GTPase domain with SRP RNA has been proposed but has never been structurally verified. In this study, we present the crystal structure at 2.5-Å resolution of the SRP54 -SRP19 -SRP RNA complex of Methanococcus jannaschii SRP. The structure reveals an RNA-bound conformation of the SRP54 GTPase domain, in which the domain is spatially well separated from the signal peptide binding site. The association of both the N and G domains with SRP RNA in free SRP provides further structural evidence for the pivotal role of SRP RNA in the regulation of the SRP54 activity.T he signal-recognition particle (SRP) targets proteins destined for membrane insertion and secretion to or across the plasma membrane in bacteria and the endoplasmic reticulum in eukaryotes. SRP binds to the ribosome as well as to the hydrophobic signal sequences of nascent polypeptide chains as they emerge from the ribosome. The ribosome-nascent chain-SRP complex is then targeted to the membrane by an interaction between SRP and its receptor (SR in eukaryotes and FtsY in bacteria). In the presence of the translocon, the signal peptide is released into the translocon channel. SRP then dissociates from SR and the ribosome-nascent chain and is ready for another cycle of cotranslational protein targeting. The targeting cycle is coordinated by GTP binding and hydrolysis by both SRP and SR (for reviews, see refs. 1-3).SRP composition varies across the three domains of life. Mammalian SRP consists of a single Ϸ300-nt RNA (SRP RNA or 7S RNA) and six proteins, which can be divided into two major functional domains: the Alu domain (comprising the proteins SRP9 and -14) and the S domain (SRP19, -54, -68, and -72). The S domain functions in signal sequence recognition and SR interaction, whereas the Alu domain is required for translational arrest on signal sequence recognition (4). Archaeal SRPs consist of a 7S RNA that is highly similar to mammalian 7S RNAs, but only two homologues of the mammalian SRP proteins, namely SRP19 and SRP54 (5). A minimal set of SRP components is found in bacteria, which consist of shorter RNAs (4.5S RNA) and only Ffh, the protein homologous to SRP54 (6, 7). SRP54, the only protein component present in all SRPs, comprises an N-terminal domain (N, a four-helix bundle), a central GTPase domain [G, a ras-like GTPase fold, with an additional unique ␣-␤-␣ insertion box domain (IBD)], and a methionine-rich C-terminal domain (M, an all-␣ structure) ...

Use of synthetic signal sequences to explore the protein export machinery

“…39,40 Recently, the role of Using Signal Peptides to Explore Protein Export 309 SRP RNA in the communication between the individual domains of Ffh (see later) and the coordination of different steps of the targeting reaction has been described. 41,42 The protein subunit present in E. coli SRP is termed Ffh, as it is the prokaryotic homolog to the fifty-four kDa subunit of the eukaryotic SRP (SRP54). Ffh (as well as SRP54) is the subunit responsible for signal sequence binding, and it is conserved in all the SRPs found in nature.…”

Domain Organization in the 54-kDa Subunit of the Chloroplast Signal Recognition Particle

“…S2). This seems reasonable in view of available crystal structures that show a large degree of variability in the positioning of the M-domain relative to the NG-domain, even in structures that contain no other binding partners such as SRP RNA or the ribosome (18)(19)(20)32,33,(60)(61)(62)(63)(64). Indeed, little conservation is observed overall for surface residues in SRP54s/ Ffhs, aside from the GTPase region (Fig.…”

Structural basis of signal peptide recognition by the signal recognition particle

“…By studying crystal structures of the SRP RNA in complex with its different protein partners, many of the structural states of the SRP have been revealed. The structures of the Methanococcus jannaschii SRP54-SRP19-S domain RNA complex in its free form [1] and in complex with an hydrophobic idealized signal peptide comprising 14 leucine and alanine residues [2] provide an explanation for how signal peptide binding at the M domain triggers a reorientation of the GTPase domain by local structuring and a-helix formation of the GM linker. …”

“…The C-terminal segment of the b-subunit wraps around the á-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the á-subunit from another ab-heterodimer. The structure of APSR, together with its oligomerization properties, suggests that APSR might self-regulate its activity through the C-terminus of the b-subunit [1, 3]. The structures of two active forms of Dsr, Dsr-I and Dsr-II, are also determined and compared.…”