Domain Interactions in E. coli SRP: Stabilization of M Domain by RNA Is Required for Effective Signal Sequence Modulation of NG Domain

Zheng, Ning; Gierasch, Lila M.

doi:10.1016/s1097-2765(00)80009-x

Cited by 75 publications

(90 citation statements)

References 33 publications

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“…Our results suggest that the binding of 4+5S RNA to Ffh not only leads to a structural change of the RNA, as discussed above, but that it also changes the structure of Ffh, although no significant structural change of the M domain fragment is seen in the crystal structure of the M domain-RNA complex (Batey et al+, 2000), compared with the M domain structure of Thermus aquaticus Ffh (Keenan et al+, 1998)+ However, the possibility remains that Ffh-RNA complex formation results in structural changes in parts of the protein that were either not present in the fragment used for crystallization, that is, the NG domain and small parts of the M domain, or that were disordered in the crystal, that is, the finger loop+ In conclusion, the present results suggest that structural details of the tetraloop region of 4+5S RNA, either of the tetraloop itself or of the adjoining stem, influence the structure of SRP such that the binding of FtsY is affected+ This implies that both RNA and Ffh undergo mutual structural adaptation upon association to form SRP+ The presumed structural change in Ffh induced by binding to 4+5S RNA probably extends beyond the M domain, which harbors the binding site for the RNA, as proteolysis data suggest that the structure of both M and NG domains of Ffh changes upon binding 4+5S RNA, indicating an influence of the RNA-binding M domain on the adjacent NG domain, as previously indicated by proteolysis experiments (Zheng & Gierasch, 1997)+ The interaction of Ffh in SRP with FtsY strongly depends on the presence of GTP (Kusters et al+, 1995;Jagath et al+, 2000), and it has been proposed that the conformational state of the G domain in response to the bound nucleotide is signaled to other domains and influences their function (Freymann et al+, 1999)+ It is likely that the modulation of the interdomain communication in Ffh, by ligands such as GTP, 4+5S RNA, or the nascent signal peptide presented by the ribosome, is an important element of the regulation of SRP function+ In this modulation, the SRP RNA appears to have a crucial role beyond that serving as a scaffold for Ffh binding+…”

Section: Discussionmentioning

confidence: 54%

Important role of the tetraloop region of 4.5S RNA in SRP binding to its receptor FtsY

Jagath

Matassova

Leeuw³

et al. 2001

RNA

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Binding of Escherichia coli signal recognition particle (SRP) to its receptor, FtsY, requires the presence of 4.5S RNA, although FtsY alone does not interact with 4.5S RNA. In this study, we report that the exchange of the GGAA tetraloop sequence in domain IV of 4.5S RNA for UUCG abolishes SRP-FtsY interaction, as determined by gel retardation and membrane targeting experiments, whereas replacements with other GNRA-type tetraloops have no effect. A number of other base exchanges in the tetraloop sequence have minor or intermediate inhibitory effects. Base pair disruptions in the stem adjacent to the tetraloop or replacement of the closing C-G base pair with G-C partially restored function of the otherwise inactive UUCG mutant. Chemical probing by hydroxyl radical cleavage of 4.5S RNA variants show that replacing GGAA with UUCG in the tetraloop sequence leads to structural changes both within the tetraloop and in the adjacent stem; the latter change is reversed upon reverting the C-G closing base pair to G-C. These results show that the SRP-FtsY interaction is strongly influenced by the structure of the tetraloop region of SRP RNA, in particular the tetraloop stem, and suggest that both SRP RNA and Ffh undergo mutual structural adaptation to form SRP that is functional in the interaction with the receptor, FtsY.

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Section: Discussionmentioning

confidence: 54%

Important role of the tetraloop region of 4.5S RNA in SRP binding to its receptor FtsY

Jagath

Matassova

Leeuw³

et al. 2001

RNA

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“…By analogy, the M-domain of cpSRP54 might preposition the NG-domain of cpSRP54. Second, the SRP RNA positions the M-domain of Ffh and allows it to transiently interact with the SRP or SR GTPase during complex formation (Zheng and Gierasch, 1997), whereas in cpSRP54 the M-domain itself is properly positioned to establish these interactions. Third, the two pathways use distinct mechanisms to stimulate complex formation.…”

Section: Discussionmentioning

confidence: 99%

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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“…The structural elements found at the N and G interface enable a dynamic communication between these domains. The C-terminal domain of Ffh, called the ''M domain'' because of its high methionine content, comprises the RNAbinding site, 47,48 although some structural studies raise the possibility that the N domain may make contact with the RNA as well. [49][50][51][52] The M domain is mostly helical and contains a highly conserved hydrophobic loop (so-called finger loop), which is disordered in some crystal structures.…”

Section: Srp Structurementioning

confidence: 99%

“…47,53 We found that the M domain is inherently flexible and binding of 4.5S RNA to Ffh stabilizes the M domain. 48 Subsequently, in a study that involved the use of fragments of the E. coli protein component of SRP, our lab found evidence that this finger loop itself is detrimental to the stability of the M domain, and proposed that one of the functional requirements FIGURE 1 Protein export to the endoplasmic reticulum (ER) and insertion into the ER membrane in eukaryotes, and to the plasma membrane or the periplasm in E. coli. SRP-mediated targeting: As a membrane protein (pink) is emerging from the ribosome (gray), SRP (light blue) recognizes and binds the signal sequence (red), and the entire nascent chain complex is targeted to the SRP receptor (dark blue) at the target membrane (yellow).…”

Section: Srp Structurementioning

confidence: 99%

“…However, other evidence showed that the NG domain is required for efficient signal peptide binding. 48,55,59 Our lab studied the interaction between signal peptides and SRP by using reconstituted fragments of SRP from E. coli and synthetic signal peptides. 48 We found that specific binding of signal peptides causes a dramatic and global destabilization of the SRP protein.…”

Section: Signal Sequence Binding By Srpmentioning

confidence: 99%

See 1 more Smart Citation

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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Domain Interactions in E. coli SRP: Stabilization of M Domain by RNA Is Required for Effective Signal Sequence Modulation of NG Domain

Cited by 75 publications

References 33 publications

Important role of the tetraloop region of 4.5S RNA in SRP binding to its receptor FtsY

Important role of the tetraloop region of 4.5S RNA in SRP binding to its receptor FtsY

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

Use of synthetic signal sequences to explore the protein export machinery

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