RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle

Spanggord, Richard J.; Siu, Fai; Ke, Ailong; Doudna, Jennifer A.

doi:10.1038/nsmb1025

Cited by 45 publications

(65 citation statements)

References 36 publications

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“…By contrast, when SRP receptor is added, no density for the NG domains of either SRP54 or the NG domain of SR␣ (the metazoan ortholog of FtsY) was observed, indicating that binding to the SR induces a conformational change (Halic et al, 2006b). Solution studies further support the notion that complex formation between the SRP and SR induce a conformational change in this region (Buskiewicz et al, 2005a;Spanggord et al, 2005). Taken together with our results, these data indicate that a specific conformation of the linker is required for GTPase stimulation by the 4.5S RNA and that factors affecting the relative conformation of the M and NG domains of Ffh could affect the rate of Ffh⅐FtsY dissociation driven by GTP hydrolysis.…”

Section: Discussionsupporting

confidence: 57%

The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting

Bradshaw

Walter

2007

MBoC

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The RNA component of the signal recognition particle (SRP) is universally required for cotranslational protein targeting. Biochemical studies have shown that SRP RNA participates in the central step of protein targeting by catalyzing the interaction of the SRP with the SRP receptor (SR). SRP RNA also accelerates GTP hydrolysis in the SRP⅐SR complex once formed. Using a reverse-genetic and biochemical analysis, we identified mutations in the E. coli SRP protein, Ffh, that abrogate the activity of the SRP RNA and cause corresponding targeting defects in vivo. The mutations in Ffh that disrupt SRP RNA activity map to regions that undergo dramatic conformational changes during the targeting reaction, suggesting that the activity of the SRP RNA is linked to the major conformational changes in the signal sequence-binding subunit of the SRP. In this way, the SRP RNA may coordinate the interaction of the SRP and the SR with ribosome recruitment and transfer to the translocon, explaining why the SRP RNA is an indispensable component of the protein targeting machinery. INTRODUCTIONCotranslational protein targeting is a major route by which proteins are targeted to the membrane of the endoplasmic reticulum (or plasma membrane in prokaryotes). The machinery required for cotranslational protein targeting consists of the signal recognition particle (SRP), a protein/RNA complex that binds ribosomes translating secretory and membrane proteins, and the SRP receptor (SR), which resides at the membrane and binds to the SRP (Keenan et al., 2001). One of the highly conserved features of the targeting machinery is that SRP requires an RNA subunit to function (Walter and Blobel, 1982). This study elaborates the mechanism by which the SRP RNA subunit contributes to the protein targeting reaction.In the first step of cotranslational protein targeting, the SRP binds to the signal sequence of a nascent polypeptide chain emerging from the ribosome (Walter et al., 1981;Keenan et al., 2001;Halic et al., 2004). The resulting SRPribosome-nascent chain complex then binds to the SRP receptor (SR), which resides at the target membrane (Gilmore et al., 1982a,b). The SRP and the SR associate with each other through related GTPase modules, but only when GTPbound (Miller et al., 1993;Egea et al., 2004;Focia et al., 2004). After transfer of the ribosome to the protein translocation channel (translocon), the SRP and the SR hydrolyze their respective bound GTPs, which causes them to dissociate, and allows for a new round of targeting (Connolly et al., 1991). The SRP and the SR reciprocally activate the other's GTPase and are therefore GTPase-activating proteins (GAPs) for each other (Powers and Walter, 1995).Although SRP-dependent protein targeting is conserved in all organisms, the prokaryotic system has the fewest components and is therefore the simplest (Poritz et al., 1990;Larsen and Zwieb, 1993). In Escherichia coli, the SRP consists of a single protein, Ffh, and a small RNA, the 4.5S RNA (Poritz et al., 1990). Ffh consists of two domains: the M d...

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Section: Discussionsupporting

confidence: 57%

The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting

Bradshaw

Walter

2007

MBoC

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“…Although models are abundant (Peluso et al, 2000;Buskiewicz et al, 2005;Spanggord et al, 2005), the molecular mechanism by which the SRP RNA acts as a catalyst to accelerate both the formation and disassembly of the SRP•SR complex is still poorly understood. It is possible that in the transition state for complex assembly, the SRP RNA may provide a transient tether that facilitates the rearrangement of one or both GTPases into the closed conformation; alternatively, the RNA and the chloroplast GTPases may use completely different mechanisms to attain a faster association kinetics.…”

Section: Discussionmentioning

confidence: 99%

Efficient Interaction between Two GTPases Allows the Chloroplast SRP Pathway to Bypass the Requirement for an SRP RNA

Jaru-Ampornpan

Chandrasekar

Shan

2007

MBoC

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Cotranslational protein targeting to membranes is regulated by two GTPases in the signal recognition particle (SRP) and the SRP receptor; association between the two GTPases is slow and is accelerated 400-fold by the SRP RNA. Intriguingly, the otherwise universally conserved SRP RNA is missing in a novel chloroplast SRP pathway. We found that even in the absence of an SRP RNA, the chloroplast SRP and receptor GTPases can interact efficiently with one another; the kinetics of interaction between the chloroplast GTPases is 400-fold faster than their bacterial homologues, and matches the rate at which the bacterial SRP and receptor interact with the help of SRP RNA. Biochemical analyses further suggest that the chloroplast SRP receptor is pre-organized in a conformation that allows optimal interaction with its binding partner, so that conformational changes during complex formation are minimized. Our results highlight intriguing differences between the classical and chloroplast SRP and SRP receptor GTPases, and help explain how the chloroplast SRP pathway can mediate efficient targeting of proteins to the thylakoid membrane in the absence of the SRP RNA, which plays an indispensable role in all the other SRP pathways.

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“…39 The importance of this role is underlined by the finding that RNA mutants unable to stimulate GTPase activity show severe growth phenotypes in in vivo complementation experiments. 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).…”

Section: Srp Structurementioning

confidence: 99%

Use of synthetic signal sequences to explore the protein export machinery

Clérico¹,

Maki²,

Gierasch³

2008

Biopolymers

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The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self‐contained, often transportable targeting sequences. Of these, signal sequences specify that a protein should be secreted from a cell or incorporated into the cytoplasmic membrane. A central puzzle is presented by the lack of primary structural homology among signal sequences, although they share common features in their sequences. Synthetic signal peptides have enabled a wide range of studies of how these “zipcodes” for protein secretion are decoded and used to target proteins to the protein machinery that facilitates their translocation across and integration into membranes. We review research on how the information in signal sequences enables their passenger proteins to be correctly and efficiently localized. Synthetic signal peptides have made possible binding and crosslinking studies to explore how selectivity is achieved in recognition by the signal sequence‐binding receptors, signal recognition particle, or SRP, which functions in all organisms, and SecA, which functions in prokaryotes and some organelles of prokaryotic origins. While progress has been made, the absence of atomic resolution structures for complexes of signal peptides and their receptors has definitely left many questions to be answered in the future. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 307–319, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle

Cited by 45 publications

References 36 publications

The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting

The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting

Efficient Interaction between Two GTPases Allows the Chloroplast SRP Pathway to Bypass the Requirement for an SRP RNA

Use of synthetic signal sequences to explore the protein export machinery

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