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. 2013 Dec 20;288(51):36385-97.
doi: 10.1074/jbc.M113.513614. Epub 2013 Oct 22.

Molecular mechanism of GTPase activation at the signal recognition particle (SRP) RNA distal end

Kuang Shen  1 , Yaqiang Wang, Yu-Hsien Hwang Fu, Qi Zhang, Juli Feigon, Shu-ou Shan
Affiliations

Molecular mechanism of GTPase activation at the signal recognition particle (SRP) RNA distal end

Kuang Shen et al. J Biol Chem. .

Abstract

The signal recognition particle (SRP) RNA is a universally conserved and essential component of the SRP that mediates the co-translational _targeting of proteins to the correct cellular membrane. During the _targeting reaction, two functional ends in the SRP RNA mediate distinct functions. Whereas the RNA tetraloop facilitates initial assembly of two GTPases between the SRP and SRP receptor, this GTPase complex subsequently relocalizes ∼100 Å to the 5',3'-distal end of the RNA, a conformation crucial for GTPase activation and cargo handover. Here we combined biochemical, single molecule, and NMR studies to investigate the molecular mechanism of this large scale conformational change. We show that two independent sites contribute to the interaction of the GTPase complex with the SRP RNA distal end. Loop E plays a crucial role in the precise positioning of the GTPase complex on these two sites by inducing a defined bend in the RNA helix and thus generating a preorganized recognition surface. GTPase docking can be uncoupled from its subsequent activation, which is mediated by conserved bases in the next internal loop. These results, combined with recent structural work, elucidate how the SRP RNA induces GTPase relocalization and activation at the end of the protein _targeting reaction.

Keywords: G Proteins; Nuclear Magnetic Resonance; Protein _targeting; Protein-Nucleic Acid Interaction; RNA; Single Molecule Biophysics.

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Figures

FIGURE 1.
FIGURE 1.
Scheme depicting the function of SRP RNA during co-translational protein _targeting. A, working model of co-translational protein _targeting by the SRP. B, secondary structure of the E. coli SRP RNA. The four sites on the RNA that mediate different functions are noted with different colors.
FIGURE 2.
FIGURE 2.
An intact SRP RNA distal end is required for efficient GTPase activation. A, GTPase assay showing the function of the SRP RNA truncation mutants. B, summary of the GTPase rate constant for single nucleotide truncation mutants of 92-mer. All kcat values are reported relative to that of wild-type SRP RNA.
FIGURE 3.
FIGURE 3.
Specific nucleotides at the SRP RNA distal end play crucial roles in GTPase activation. A–D, site-directed mutagenesis of specific bases at the SRP RNA distal end and their GTPase activity. The four nucleotides whose mutations cause the most deleterious effects are shown: G14 (A), U15 (B), G96 (C), and U98 (D). E, summary of the GTPase activity of the point mutants at the distal end docking site. Values in parentheses denote the kcat values of the mutant relative to that of wild-type RNA. F, GTPase activity of base pair-switched mutants.
FIGURE 4.
FIGURE 4.
Catalytic bases in loop D specifically contribute to catalysis. A, crystal structure of the SRP-FtsY complex in which the GTPase complex is docked at the distal end (Protein Data Bank ID 2XXA (27). Shown in yellow is the protruding base (C86) that inserts into the Ffh-FtsY NG domain interface. B, secondary structure of the SRP RNA loop D. C, single molecule setup to observe the migration of the Ffh-FtsY NG complex along the SRP RNA. Ffh-C153 is labeled with Cy3. The 3′-end of the SRP RNA is labeled with Quasar670. D, fluorescence signals (upper panel) and FRET trajectory (lower panel) of the SRP-FtsY complex in GppNHp. HMM of the FRET trajectory is shown in navy. E, single molecule traces (left panel) and FRET histograms (right panel) of G83A and C86G mutants.
FIGURE 5.
FIGURE 5.
Correlation between the probability of attaining the high FRET state and the observed GTPase activity of the SRP-FtsY complex. Standard curve (dashed line in A and solid line in B) is the linear fit of the six data points with WT RNA and distal site docking mutants (B). The data for G99A and 82mer RNA are from Ref. and were included in the linear regression. The data points for G83A, C86G, and C87A (colored circles) are not included in the fit.
FIGURE 6.
FIGURE 6.
C87 provides an auxiliary docking site for the GTPase complex. A, GTPase activity of C87 mutants. B, single molecule trace (left panel) and FRET histogram (right panel) of the C87A mutant. C, C87 acting independently of the distal docking site. GTPase activity of the SRP RNA contains a combination of activating mutations. C97U and G99A are activating mutations at the primary docking site. C87A is the activating mutation at the auxiliary docking site.
FIGURE 7.
FIGURE 7.
Loop E plays a crucial role in GTPase activation by the SRP RNA. A, loop E mutants characterized in this work. E−1, E+1, and E+2 alter the size of loop E. Ecg reduces potential dynamics of loop E by replacing the UA pairs with CG pairs. ΔE, ΔE+1, and cE eliminate loop E. B, GTPase activity of the loop E mutants in A. C, summary of the relative GTPase rate constant (kcat) of the loop E mutants relative to wild-type RNA.
FIGURE 8.
FIGURE 8.
Loop E mutants disrupt correct docking of the GTPase complex at the distal end of the SRP RNA. Sample FRET trajectories (cyan) and HMM simulation (navy) of E−1, E+1, and Ecg SRP RNA mutants (left panel) are shown. The FRET histograms for each mutant are shown in the right panel.
FIGURE 9.
FIGURE 9.
NMR study directly visualizes the orientation and flexibility of loop E region. A, sequence and secondary structures of WTx, E+1x, and E−1x constructs. B, schematic representation of the secondary structures of WT, E+1, and E−1 derived from NMR data. C, computational modeling of the NMR-derived structures of WT (green), E+1 (red), and E−1 (blue) superimposed on ED stem. G14, U15, G96, and U98 are colored magenta. The elongated gray nucleotides on ED stem are guides for visualization. D, RDC analysis results of WTx, E+1x, and E−1x.
FIGURE 10.
FIGURE 10.
Comparison of elongated WT SPR (WTx) and WT SRP. A, sequence and secondary structures of WT SPR and WTx constructs. B–D, overlapping spectra of two-dimensional 1H-13C HSQC of the C6H6/C8H8 (B), sugar C1′H1′ (C), and C5H5 (D) from WT (orange) and WTx (green). The asterisks denote resonances that belong to two terminal guanine (G−21 and G−22) and cytosine (C+21 and C+22) residues in WTx.
FIGURE 11.
FIGURE 11.
Identification of base pairing of the wild-type SRP RNA by NMR spectra. A, two-dimensional NOESY of wild-type SRP RNA in H2O. Selected assignments are labeled in the spectrum. The NOEs (C17H41-G93H1 and C17H42-G93H1) provide direct evidence that C17 and G93 form a canonical GC base pair. B, imino-imino region in two-dimensional NOESY of WT SRP RNA in H2O. The lines indicate sequential imino proton connectivities. The inset in B shows the sequence and secondary structure of wild-type SRP RNA. The assignments and lines are colored by subdomain as in the inset.
FIGURE 12.
FIGURE 12.
1H-15N HSQC spectra of WTx (A), E+1x (B), and E−1x (C). The cross-peaks represent base-paired guanines. Base-paired G93 can be observed in WTx, but not in E+1x and E−1x.
FIGURE 13.
FIGURE 13.
Identification of base stacking of the wild-type SRP RNA by two-dimensional NOESY in D2O. The cross-peaks are between the neighboring aromatic protons (H2, H6, or H8). No NOE connecting with U94H6 was observed. NOE (G93H8-A95H8) indicates that the base of G93 is stacked on A95 and U94 is flipped out of loop E. The assignments are colored by subdomain as in the inset of Fig. 11B.
FIGURE 14.
FIGURE 14.
Normalized resonance intensities of WT (A), E+1 (B), and E−1 (C) from non-constant-time two-dimensional 1H-13C HSQC experiments. The nucleotides are colored by subdomain as in the inset of Fig. 11B.
FIGURE 15.
FIGURE 15.
Bidentate interaction between the Ffh-FtsY GTPase complex and the distal end of the SRP RNA (Protein Data Bank ID 4C7O) (48). The four critical nucleotides that form the minor groove in the primary docking site are shown in orange. C87, the protruding base that forms the auxiliary docking site, is in magenta. The catalytic base G83 is in red.
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References

    1. Akopian D., Shen K., Zhang X., Shan S. (2013) Signal recognition particle: an essential protein-_targeting machine. Annu. Rev. Biochem. 82, 693–721 - PMC - PubMed
    1. Keenan R. J., Freymann D. M., Stroud R. M., Walter P. (2001) The signal recognition particle. Annu. Rev. Biochem. 70, 755–775 - PubMed
    1. Walter P., Johnson A. E. (1994) Signal sequence recognition and protein _targeting to the endoplasmic reticulum membrane. Annu. Rev. Cell Biol. 10, 87–119 - PubMed
    1. Halic M., Becker T., Pool M. R., Spahn C. M., Grassucci R. A., Frank J., Beckmann R. (2004) Structure of the signal recognition particle interacting with the elongation-arrested ribosome. Nature 427, 808–814 - PubMed
    1. Halic M., Blau M., Becker T., Mielke T., Pool M. R., Wild K., Sinning I., Beckmann R. (2006) Following the signal sequence from ribosomal tunnel exit to signal recognition particle. Nature 444, 507–511 - PubMed

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