1. Domain Interactions in E
Domain Interactions in E. coli SRP: Stabilization of M Domain by RNA Is Required for Effective Signal Sequence Modulation of NG Domain 
Ning Zheng, Lila M Gierasch 
Molecular Cell 
Volume 1, Issue 1, Pages (December 1997)
DOI: /S (00)80009-X

2. Figure 1
The Ffh M Domain Has a Significantly Higher Protease Susceptibility than the NG Domain
(A) Time course of V8 digestion of Ffh. The digestion reaction (Ffh:V8 = 100:1 w/w), as described in the Experimental Procedures, was stopped at the indicated time point and analyzed on SDS-PAGE. Proteolytically generated M domain was digested at a much greater rate than the NG domain.
(B) Overexpressed His-tagged Ffh M domain was treated with V8 protease (Ffh:V8 = 100:1 w/w) at room temperature under the same conditions as (A).
Molecular Cell 1997 1, 79-87DOI: ( /S (00)80009-X)

3. Figure 2 Functional Ffh M Domain Has “Molten-Globule”–like Features
(A) Overexpressed Ffh and the Ffh M domain have the same 4.5S RNA binding affinity (∼5 nM) measured by a filter binding assay.
(B) The CD spectrum of the isolated Ffh M domain shows a predominantly α-helical structure. The ellipticity minima at 208 nm and 222 nm are characteristic of α helix.
(C) Thermal denaturation of the Ffh M domain was monitored by CD at 222 nm. The unfolding of the Ffh M domain represents a noncooperative process in contrast to the transition observed in intact Ffh (see Figure 5F).
Molecular Cell 1997 1, 79-87DOI: ( /S (00)80009-X)

4. Figure 5
Effects on the Stability of Ffh and the NG Domain by Binding of Signal Peptides
(A) Ffh (1 μM) was mixed with 100 μM KRRnoW and then treated with V8 protease (right half) as described in the Experimental Procedures. On the left, no KRRnoW was included.
(B) Titration of KRRnoW in the V8 protease digestion assay of Ffh. 5 μM KRRnoW is enough for half maximum effect of enhanced proteolytic lability of Ffh (1 μM).
(C) Nonfunctional signal peptide KRR13W14D was titrated in the digestion assay with Ffh. At all the concentrations examined, no effect on the protease accessibility of Ffh was observed.
(D) Ffh (1 μM) was mixed with 20 μM OmpA signal peptide and then treated with V8 protease as described in the Experimental Procedures. Similar dramatically increased protease susceptibility of Ffh was observed.
(E) The CD spectra of 4 μM Ffh (1) and 40 μM KRRnoW (2), both in 10 mM NaHEPES (pH 7.6) at 25°C, were taken individually and then summed. The resulted spectrum (4, dotted) is compared with the spectrum of the mixture of 4 μM Ffh and 40 μM KRRnoW (3).
(F) The thermal denaturation profiles of 4 μM Ffh and 40 μM KRRnoW (upper panel), both in 10 mM NaHEPES (pH 7.6), were obtained by monitoring the ellipticity changes at 222 nm. The sum of these two profiles results in a highly cooperative curve (lower panel, dotted), in contrast to the flat melting curve of the mixture of 4 μM Ffh and 40 μM KRRnoW (lower panel).
(G–H) Ffh (4 mg/ml) was treated with V8 protease (Ffh:V8 = 50:1) as in Figure 1A. The V8 protease was less active in this specific batch yet provided a convenient “marker” for its existence. After 1 hr digestion, the sample is diluted from 4 mg/ml to 0.05 mg/ml (i.e., 1 μM) (total Ffh) by the same digestion buffer system and transferred to ice. The digested sample was then passed through cation exchange CM resin equilibrated with the same buffer and the supernatant was collected. This step removes any undigested Ffh and the Ffh M domain. Subsequently, the sample is shifted to 25°C and the digestion is continued from 1 hr. Aliquots of the sample were taken at indicated time point for SDS-PAGE analysis. (H) Same as (G), except KRRnoW (left panel) and K5W(AL)10 (right panel) were added to the sample after it was passed through the CM resin. The final concentrations of KRRnoW and K5W(AL)10 were 20 μM and 10 μM, respectively.
Molecular Cell 1997 1, 79-87DOI: ( /S (00)80009-X)

5. Figure 3
4.5S RNA and Its Domain IV Fragment Stabilize the Ffh M Domain
(A) Ffh was incubated with equimolar amount of 4.5S RNA at room temperature for 10 min and then treated with V8 protease as described in methods. Proteolytically generated M domain stayed intact as did the NG domain.
(B) V8 protease digestion of Ffh, with or without 4.5S RNA dIV, indicates that the domain IV region of 4.5S RNA is sufficient for M domain stabilization.
(C) Comparison of the thermal denaturation curves of the Ffh M domain alone and the Ffh M domain in complex with 4.5S RNA dIV (bottom) shows a clear transition in the presence of the RNA. The thermal denaturation profile was obtained by monitoring the CD signal of the samples at 222 nm.
(D) Thermal denaturation of 4.5S RNA dIV alone monitored at 222 nm (top). The near zero ellipticity of RNA at 222 nm did not change as temperature increased. CD spectra of 4.5S RNA dIV alone and Ffh M domain/4.5S RNA dIV complex. As 4.5S RNA shows near zero ellipticity at 222 nm, the helical Ffh M domain contributes a characteristic negative peak.
Molecular Cell 1997 1, 79-87DOI: ( /S (00)80009-X)

6. Figure 4
Inhibition of Ffh GTPase Activity by Synthetic Signal Peptides
(A) GTP hydrolysis by Ffh was monitored following the previously published methods (Miller et al. 1994). The Ffh concentration in the 100 μl reaction was 100 nM. No 4.5S RNA was added. KRRnoW shows similar inhibitory activity as wild-type LamB peptide whereas nonfunctional KRR13W14D peptide has negligible effect on the GTP hydrolysis.
(B) Comparison of the peptide concentration dependence of the Ffh GTPase inhibition in the presence and absence of 4.5S RNA. Whereas the basal GTPase activity of Ffh is slightly increased upon binding to 4.5S RNA, KRRnoW half inhibits the GTP hydrolysis at a similar concentration both with and without 4.5S RNA.
Molecular Cell 1997 1, 79-87DOI: ( /S (00)80009-X)

7. Figure 6
4.5S RNA Inhibits the Globally Destabilizing Effect of Signal Peptides on Ffh
In lane 2, Ffh alone was treated with V8 protease. In lane 3, Ffh was incubated with 4.5S RNA first and then treated with V8 protease. KRRnoW (20 μM) was added to Ffh solution in lane 4 to 6. No 4.5S RNA was added in lane S RNA was added first in lane 5 whereas it was added 30 min after the addition of KRRnoW in lane 6.
Molecular Cell 1997 1, 79-87DOI: ( /S (00)80009-X)

8. Figure 7
A Model for the Essential Structural and Functional Roles Played by 4.5S RNA in E. coli SRP
In the absence of 4.5S RNA, the Ffh M domain is in a molten globule–like state (disorganized helices and broken outline) in contrast to the well-folded NG domain. Upon binding to a signal peptide, although peptide-binding induces domain contact, both NG and M domains (fuzzy filling and no outline) become highly unstable because of an improper interface. RNA binding to the M domain, which stabilizes the polypeptide and results in a more specific structure (organized helices), establishes the correct communication between the two domains. This domain–domain interaction stabilizes the NG domain under the influence of signal sequence binding. See text for more discussion. The final complex of Ffh/4.5S RNA/signal peptide is functionally competent to interact with FtsY on the membrane.
Molecular Cell 1997 1, 79-87DOI: ( /S (00)80009-X)