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

Bradshaw, Niels; Walter, Peter

doi:10.1091/mbc.e07-02-0117

Cited by 32 publications

(42 citation statements)

References 40 publications

(69 reference statements)

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“…Both these RNA activities may be modulated by the presence of the signal sequence and the ribosome (19,(41)(42)(43)(44)(45). A recent model of the E. coli SRP-FtsY complex suggests that the SRP RNA localizes at the Ffh-FtsY heterodimer interface on SRP-FtsY association (19).…”

Section: Discussionmentioning

confidence: 99%

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

Hainzl

Huang

Sauer‐Eriksson

2007

Proc. Natl. Acad. Sci. U.S.A.

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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) ...

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Section: Discussionmentioning

confidence: 99%

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

Hainzl

Huang

Sauer‐Eriksson

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…The stimulatory effect of the cpSRP54 M-domain is most intriguing because E. coli Ffh exhibits similar interaction kinetics with FtsY regardless of whether its M-domain is present ( Figure 3B; Bradshaw and Walter, 2007;Chandrasekar et al, 2008). The interaction between the E. coli GTPases is only stimulated when the M-domain binds the SRP RNA ( Figure 3B; Peluso et al, 2001).…”

Section: The M-domain Of Cpsrp54 Accelerates Cpsrp54 -Cpftsy Associationmentioning

confidence: 99%

“…The faster k cat /K m value in the presence of cpSRP54 Mdomain implies that the M-domain accelerates the kinetics of (Chandrasekar et al, 2008;Bradshaw and Walter, 2007) remaining in the monomer form ( Figure 4A, red). In contrast, complex formation is much slower in the case of cpSRP54 NG ( Figure 4B).…”

Section: The M-domain Of Cpsrp54 Accelerates Cpsrp54 -Cpftsy Associationmentioning

confidence: 99%

“…Moreover, the SRP RNA accelerates the rate at which the SRP • SR complex hydrolyzes GTP 5-to 10-fold (Peluso et al, 2001;Siu et al, 2007). Many reports have suggested that the SRP RNA may play a regulatory role by bridging the communication between cargo binding and the GTPase cycle (Batey et al, 2000;Bradshaw and Walter, 2007;Bradshaw et al, 2009). The SRP RNA therefore plays a crucial role in the SRP pathway, explaining why it is highly conserved from bacteria to archaea to eukaryotes.…”

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confidence: 99%

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A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

Jaru-Ampornpan

Nguyen

Shan

2009

MBoC

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Cotranslational protein targeting by the signal recognition particle (SRP) requires the SRP RNA, which accelerates the interaction between the SRP and SRP receptor 200-fold. This otherwise universally conserved SRP RNA is missing in the chloroplast SRP (cpSRP) pathway. Instead, the cpSRP and cpSRP receptor (cpFtsY) by themselves can interact 200-fold faster than their bacterial homologues. Here, cross-complementation analyses revealed the molecular origin underlying their efficient interaction. We found that cpFtsY is 5-to 10-fold more efficient than Escherichia coli FtsY at interacting with the GTPase domain of SRP from both chloroplast and bacteria, suggesting that cpFtsY is preorganized into a conformation more conducive to complex formation. Furthermore, the cargo-binding M-domain of cpSRP provides an additional 100-fold acceleration for the interaction between the chloroplast GTPases, functionally mimicking the effect of the SRP RNA in the cotranslational targeting pathway. The stimulatory effect of the SRP RNA or the M-domain of cpSRP is specific to the homologous SRP receptor in each pathway. These results strongly suggest that the M-domain of SRP actively communicates with the SRP and SR GTPases and that the cytosolic and chloroplast SRP pathways have evolved distinct molecular mechanisms (RNA vs. protein) to mediate this communication. INTRODUCTIONThe signal recognition particle (SRP) and the SRP receptor (SR) comprise the major cellular machineries that cotranslationally deliver newly synthesized proteins from the cytosol to target membranes (Walter and Johnson, 1994;Keenan et al., 2001). Cotranslational protein targeting begins with recognition of the cargo-ribosomes translating nascent polypeptides containing signal sequences-by the SRP (Walter et al., 1981). The cargo is brought to the vicinity of the target membrane via the interaction between the SRP and SRP receptor (FtsY in bacteria) (Gilmore et al., 1982a). On arrival at the membrane, SRP unloads its cargo to the protein-conducting channel, composed of the sec61p complex in eukaryotic cells or secYEG complex in bacteria and archaea (Simon and Blobel, 1991;Gorlich et al., 1992;Halic et al., 2006). The SRP and SRP receptor also reciprocally stimulate each other's GTPase activity (Powers and Walter, 1995). Thus, after cargo unloading, guanosine triphosphate (GTP) hydrolysis drives disassembly of the SRP • SR complex, returning the components into the cytosol for the next round of protein targeting (Connolly et al., 1991).The SRP pathway is conserved throughout all three kingdoms of life. Although the protein components of SRP and SR vary across species, the functional core of SRP is a highly conserved ribonucleoprotein complex, composed of a 54-kDa SRP GTPase (SRP54 in eukaryotes or Ffh in bacteria) and an SRP RNA (Keenan et al., 2001). The SRP receptor also contains a conserved GTPase domain that is highly homologous to the GTPase domain in SRP54, and together the GTPase domains of SRP and SR form a unique subgroup in the GTPase superfamily (Keen...

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“…Ffh recognizes and binds to the secretion signal sequences of secreted or transmembrane proteins as they emerge from the ribosome. In a process that is accelerated by the 4.5S RNA, the Ffh-nascent polypeptide complex binds the membrane receptor filament temperature-sensitive Y (FtsY), which delivers the nascent polypeptide to the Sec translocation machinery (Bradshaw & Walter, 2007). FfhFtsY complex formation also stimulates the GTPase activities of both proteins, leading to their dissociation and making them available for additional rounds of protein targeting (Powers & Walter, 1995).…”

Section: Introductionmentioning

confidence: 99%

The 4.5S RNA component of the signal recognition particle is required for group A Streptococcus virulence

2010

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The signal recognition particle (SRP) is a ribonucleoprotein complex that targets proteins for secretion in a co-translational manner. While originally thought to be essential in all bacteria, recent data show that the SRP is dispensable in at least some streptococcal species. The SRP from the human pathogen group A Streptococcus (GAS, Streptococcus pyogenes) is predicted to be composed of protein Ffh and 4.5S RNA. Deletion of ffh alters the secretion of several GAS proteins, and leads to a severe reduction in virulence. Here, we report that mutation of the gene encoding 4.5S RNA results in phenotypes both similar to and distinct from that observed following ffh mutation. Similarities include a reduction in secretion of the haemolysin streptolysin O, and attenuation of virulence as assessed by a murine soft tissue infection model. Differences include a reduction in transcript levels for the genes encoding streptolysin O and NADglycohydrolase, and the reduced secretion of the SpeB protease. Several differences in transcript abundance between the parental and mutant strain were shown to be dependent on the sensorkinase-encoding gene covS. Using growth in human saliva as an ex vivo model of upper respiratory tract infection we identified that 4.5S RNA mutation leads to a 10-fold reduction in colony-forming units over time, consistent with the 4.5S RNA contributing to GAS growth and persistence during upper respiratory tract infections. Finally, we determined that the 4.5S RNA was essential for GAS to cause lethal infections in a murine bacteraemia model of infection. The data presented extend our knowledge of the contribution of the SRP to the virulence of an important Gram-positive pathogen.

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

Cited by 32 publications

References 40 publications

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

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

A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

The 4.5S RNA component of the signal recognition particle is required for group A Streptococcus virulence

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