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Visualizing induced fit in
early assembly of the
human signal recognition
particle
structure essential for further assembly, in this case with SRP54.
Using a single-nucleotide resolution footprinting approach, we
identify three distinct steps in assembly of the SRP19–RNA complex. First, an initial unstable, but structurally specific, encounter
complex forms. Then, assembly to the native complex involves two
subsequent processes of structural consolidation to create the RNA
structure compatible with high affinity binding by SRP54.
Marsha A. Rose and Kevin M. Weeks
SRP19–RNA assembly, disassembly and binding affinity
To determine the association rates, SRP19 was added to radiolabeled LS RNA to initiate complex formation (Fig. 2a). The association rate constant (kon) is (7 ± 2) × 107 M–1 min–1 (Fig. 2b),
which is significantly slower than observed for protein–RNA
association reactions involving components whose tertiary
structure is preformed17 or than expected for diffusion limited
assembly (∼109 M–1 min–1). Complex disassembly (koff) was
monitored by preforming an SRP19–LS RNA complex and subsequently trapping dissociated protein using either 200-fold
dilution or addition of excess unlabeled competitor RNA. Both
approaches yielded similar dissociation rate constants of 1 ×
10–4 min–1 (Fig. 2c), corresponding to a half life of 120 hours.
Because SRP19–RNA complex dissociation is slow, care was
required when measuring the equilibrium dissociation constant
(Kd). We performed nitrocellulose filter binding experiments in
which the LS RNA concentration was held to a low (relative to
Kd) concentration of 0.01 nM and equilibrated with SRP19 for
long times. The Kd is 2 nM for equilibration periods spanning
2–120 hr (Fig. 2d). To verify that the SRP19–LS RNA complex
scored in our assays represents formation of a specific ribonucleoprotein complex, we performed binding experiments with an
RNA variant containing an A149U point mutation that disrupts
SRP19 binding (ref. 18) (Fig. 1). No SRP19–A149U LS RNA
complex was observed at protein concentrations <20 µM
(Fig. 2d, closed triangles).
Department of Chemistry, University of North Carolina, Chapel Hill,
North Carolina 27599-3290, USA.
Assembly of almost all ribonucleoprotein complexes
involves induced fit in the RNA and, thus, formation of one
or more intermediate states. In assembly of the human signal recognition particle (SRP), we show that SRP19 binding
to SRP RNA involves obligatory intermediates. An apparent
discrepancy exists between the ratio of dissociation and
association rate constants, determined in a partitioning
experiment, and the equilibrium binding constant; this
kinetic signature reflects formation of a stable intermediate
in assembly of the ribonucleoprotein complex. Assembly
intermediates were observed directly by time-resolved footprinting. SRP19 binds rapidly to SRP RNA to form an initial
labile, but structurally specific, encounter complex involving both helices III and IV. Two subsequent steps of structural consolidation yield the native RNA–protein interface.
SRP19 binding stabilizes helix IV in the region recognized
by SRP54, consistent with protein–protein cooperativity
mediated in part by mutual recognition of similar RNA
structures. This mechanism illustrates principles general to
ribonucleoprotein assembly reactions that rely on recruitment of architectural RNA binding proteins.
Assembly of RNA–protein complexes usually proceeds via
induced fit in the RNA or in both RNA and protein compo- An obligatory intermediate
nents1–3. Understanding the mechanisms and intervening states by If the stable SRP19–RNA complex forms in a single step, the Kd
which architectural RNA binding proteins initially recognize and could be calculated as the ratio of koff to kon and compared directsubsequently assemble with their cognate RNAs is
a challenging problem. In one example, assembly
of the human signal recognition particle (SRP)
yields a ribonucleoprotein comprised of two
structural units: the Alu and large subunits
(Fig. 1). The SRP9 and SRP14 proteins and ∼1/2
of the SRP RNA form the Alu subunit, whereas
SRP19, SRP54 and a SRP68–SRP72 heterodimer
bind to the RNA and comprise the large subunit4–7. SRP then targets the ribosomal complex
containing a nascent polypeptide to the endoplasmic reticulum4,8,9.
SRP19 binds to helices III and IV in the large
subunit (LS) RNA with the same affinity as to
intact full-length RNA10–13. Prior assembly of
SRP19 with the RNA strengthens binding of SRP54
to the SRP RNA13–16. Thus, assembly of the
SRP19–RNA complex represents an important
example in which induced fit binding by an architectural protein creates a new ribonucleoprotein
Fig. 1 Secondary structures of SRP RNA and the large
subunit (LS) RNA. Helical regions are identified with
Roman numerals21; other numbering systems are also
in use22. LS RNA is numbered relative to the intact SRP
RNA.
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a
b
c
d
ly to the Kd determined at equilibrium. The ratio koff / kon for the
SRP19–RNA interaction (1 × 10–4 min–1 / 7 × 107 M–1 min–1)
equals 1 pM and differs from the equilibrium Kd of 2 nM
(Fig. 2d) by three orders of magnitude. If an RNA–protein intermediate complex and the final complex are both stable to nitrocellulose partitioning, then kon and koff report binding and
disassembly at different elementary steps, and their ratio need
not equal the equilibrium Kd. Such intermediates are the essential elements of any induced fit assembly mechanism.
Mapping SRP19 binding and RNA conformational changes
We characterized structural changes in the SRP RNA upon addition of SRP19 using two complementary approaches: dimethyl
sulfate (DMS) modification and hydroxyl radical cleavage. DMS
methylation reports interactions at N1 in the Watson-Crick base
pairing face of adenine positions19, whereas hydroxyl radical
516
Fig. 2 Assembly, disassembly and equilibrium binding of the SRP19–RNA
complex. a, Assembly of the SRP19–RNA complex was monitored by
nitrocellulose filter binding at the indicated concentrations of SRP19.
Rates were obtained by fitting the fraction bound to an equation
describing a single (concentrations 1–3 nM) or double exponential (concentrations >3 nM; kon was determined using the fast phases). b, Rates
obtained in individual binding experiments are plotted as a function of
SRP19 concentration; the slope yields a kon of (7 ± 2) × 107 M–1 min–1.
c, Disassembly of a SRP19-radiolabeled RNA complex initiated by two
independent methods: 200-fold dilution (circles) or addition of 5,000fold excess unlabeled RNA (squares). The dissociation rate (koff) is 1 ×
10–4 min–1. The estimated upper limit for this rate is 4 × 10–4 min–1.
d, Binding affinity of the SRP19–RNA complex was determined by coretention of LS RNA on a nitrocellulose filter. To ensure that measurements
report equilibrium, individual reactions were filtered at 2 (circles), 24
(squares) and 120 h (diamonds). Fraction bound was normalized to the
value observed at 100 nM; fraction bound decreased from 0.6 at 2 h to
0.2 at 120 h, presumably due to RNA degradation. Curves were fit to the
Langmuir isotherm; the Kd was 2 nM in all cases. The A149U mutant does
not form a complex with SRP19 stable to nitrocellulose partitioning
under these conditions (closed triangles).
cleavage detects the solvent accessibility of the phosphoribose
backbone20. Changes in the local environment of adenosine
residues were mapped by comparing the DMS reactivity of free
LS RNA with that of a preformed SRP19–RNA complex. Most
adenosine residues in the LS RNA are methylated by DMS in the
absence of SRP19 (compare denaturing and –SRP19 lanes,
Fig. 3a), indicating that most adenines do not form canonical
base pairs (Fig. 1). Upon addition of SRP19, adenosine residues
in both helices III and IV become protected from DMS modification (representative data for helix IV are shown in Fig. 3a).
DMS footprinting supports a model in which SRP19 binding
stabilizes the RNA conformation required for recognition by
SRP54 (Fig. 3c). Upon SRP19 binding, the DMS protection pattern in the conserved symmetric internal loop in helix IV
(refs 21,22) corresponds closely to that expected for the RNA
conformation recognized by the bacterial homolog of SRP54,
Ffh23. In the absence of SRP19, all three adenine bases in this
region react efficiently with DMS (Fig. 3a). Upon SRP19 addition, A205 becomes protected from alkylation, whereas A192
and A208 remain reactive (Fig. 3c). This pattern is consistent
with the structure of the Ffh M-domain–RNA complex23 because
A205 forms an A–G pair in the Ffh–RNA complex, whereas A192
and A208 form A–C mismatches involving the non-WatsonCrick face of the adenine bases, in which the N1 position would
be accessible in the absence of a Ffh/SRP54 protein.
In the absence of SRP19, the entire RNA backbone was relatively reactive towards cleavage by the hydroxyl radical (Fig. 3b,
–SRP19 lane). We observed some modulation of the cleavage
intensity in the absence of SRP19, both under our standard binding conditions and in the absence of added monovalent or divalent ion, suggesting partial prior RNA tertiary structure
formation. Many RNA structures become less accessible towards
hydroxyl radical cleavage upon SRP19 binding (compare – and
+ SRP19 lanes, Fig. 3b).
Regions protected from DMS modification and from hydroxyl
radical cleavage (Fig. 3c) extend from the junction linking helices
II, III and IV to the loops at the termini of helices III and IV. A
similar footprint for SRP19 is observed in the case of an archaeal
SRP complex13. These data emphasize that substantial new
RNA–protein and RNA–RNA interfaces form during assembly.
Visualizing induced fit by time-resolved footprinting
Since assembly of the SRP19–LS RNA complex occurs on the
minute time scale at nanomolar concentrations (Fig. 2b), we
required a time-resolved method with nucleotide resolution but
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Fig. 3 Visualization of SRP19–RNA complex formation by DMS and hydroxyl radical footprinting. a, LS
RNA was treated with DMS under denaturing conditions or in the absence (–) or presence (+) of SRP19.
Adenines protected from methylation upon SRP19
binding are identified with triangles. Modification
was detected as stops to primer extension by reverse
transcriptase. A and G marker lanes were generated
in dideoxy sequencing reactions. Prequench indicates
that the stop solution was added prior to DMS.
b, Hydroxyl radical footprinting of LS RNA in the
absence (–) and presence (+) of SRP19. Bars denote
regions protected upon SRP19 binding. A and G
markers were generated by iodine cleavage of phosphorothioate-substituted RNA. c, Nucleotides protected from DMS (closed circles) and hydroxyl radical
cleavage (boxes) upon SRP19 binding are mapped
onto the secondary structure of LS RNA (structure of
domain IV adapted from ref. 23). Adenines accessible
to DMS in the SRP19–LS RNA complex are indicated
with open circles. Arrow brackets identify the RNA
regions monitored in these experiments.
a
only modest time resolution. An excellent
approach proved to be iodine-mediated cleavage
of RNAs partially substituted with Rp phosphorothioate linkages24,25. We used a static footprinting approach to monitor changes in reactivity of c
phosphorothioate substitutions upon SRP19
binding using the same rapid 10 s reaction and
quench procedure that would be subsequently
used in time-resolved experiments. Unreacted
iodine was neutralized by addition of 2-mercaptoethanol. Upon binding by SRP19, large regions
of the RNA are protected from cleavage by iodine
(Fig. 4a, compare – and + SRP19 lanes; both
open and closed boxes denote protection).
Protected regions span all three helical regions of
the LS RNA (Fig. 4f, boxed regions) and, as
expected, are similar but not identical to the
DMS and hydroxyl radical footprints (Fig. 3c).
An interference binding experiment performed
under conditions of limiting protein showed that
none of the Rp phosphorothioate substitutions
affected the affinity of the SRP19–RNA complex (data not shown).
We will show in a subsequent section that assembly of the
SRP19–LS RNA complex proceeds via a rapidly formed, labile
encounter complex. We mapped potential interactions in this
encounter complex by footprinting the A149U mutant (Fig. 1) at
40 nM SRP19. SRP19 does not form a complex with the A149U
RNA that is stable to nitrocellulose partitioning at this protein
concentration (Fig. 2d). However, we observed a compact footprint for the protein–A149U RNA complex spanning a subset of
residues protected in the native complex (Fig. 4a, closed boxes;
representative data for the guanosine-substituted pool is shown
explicitly). Protection is centered at the double C–A mismatch in
helix III and at a compact trinucleotide region in domain IV
(Fig. 4f, heavy boxes).
Time-resolved ribonucleoprotein assembly
Time-resolved footprinting experiments were performed at 20
and 40 nM SRP19. Stable complexes at these protein concentrations form at 1.4 and 2.8 min–1, respectively (Fig. 2b). Since
formation of an RNA–protein complex is a bimolecular reaction,
a subset of positions is expected to be protected from iodinemediated cleavage in a protein concentration-dependent manner. In contrast, formation and rearrangement of intermediates
might occur at rates independent of protein concentration.
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b
Positions A149, A199, A205 and G206 exhibited protection
profiles that varied with SRP19 concentration. Upon SRP19
binding, reactivity at A149, A199 and A205 is reduced, whereas
reactivity at G206 is enhanced. Positions A149 and A199 were
protected from iodine cleavage at 20 and 40 nM SRP19 at rates of
1.4 and 3.1 min–1, respectively (data for A149 is shown by open
and closed circles in Fig. 4b). These rates are the same, within
error, to those for stable complex formation (Fig. 2b). Protection
at positions A205 and G206 was also protein concentrationdependent but distinct from the protection observed for A149
and A199. At 20 nM protein, protection was characterized by a
reproducible lag. However, at 40 nM concentrations, protection
proceeded via a single exponential characterized by a slow rate of
0.8 min–1 (Fig. 4b, open and closed squares, respectively).
A lag occurs because there are two kinetically significant steps
with similar rates such that an intermediate must build up prior
to formation of the final complex (Fig. 4b, open squares). When
the rate of one of the steps is accelerated (by increasing the protein concentration), a single, slow step is observed (Fig. 4b, solid
squares).
Independent evidence for this slow step comes from the second observed pattern of protection. Many positions are protected more slowly (0.3–0.8 min–1) than stable complex
formation (3 min–1 at 40 nM SRP19; representative data for
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C151 and A201 are shown in Fig. 4c; green residues in Fig. 4f).
Since protection occurred at the same slow rates at both protein
concentrations, folding at these positions represents a unimolecular consolidation to the native complex. Also possibly in this
class are three positions, G153, A157 and A160, that are protected at a rate of ∼2 min–1, independent of SRP19 concentration (Fig. 4d).
Finally, a third class of positions, spanning helices III and IV, is
completely protected from iodine-mediated cleavage prior to
a
taking the first time point at 20 s at both SRP19 concentrations
tested (representative data for G156 and C189 are shown in
Fig. 4e; Fig. 4f, red regions). This assembly event occurs significantly faster than the stable complex formation observed by
nitrocellulose filter binding. Strikingly, the positions protected
from iodine-mediated cleavage more rapidly than the stable
complex can form are exactly those that footprint in the A149U
mutant (Fig. 4f, compare heavy boxed regions with red positions).
b
c
d
e
f
Fig. 4 Time-resolved assembly of the SRP19–RNA complex. a, Protection from iodine cleavage upon SRP19 binding (+) was compared to the reactivity of the free RNA (–) for each phosphorothioate-substituted nucleotide (A, G, C and U). Marker lanes (M) were cleaved under denaturing conditions.
A complex of SRP19 with the A149U RNA variant was also footprinted, and representative data for guanosine is shown. Solid boxes indicate positions
protected in both wild type and A149U mutant RNAs; open boxes identify nucleotides protected in the native complex only. The open triangle identifies one position that exhibits increased reactivity in the presence of SRP19. Prequench indicates reactions in which stop solution was added prior to
iodine. Time resolved footprinting of: b, A subset of positions, including A149 (circles) and G206 (squares) that show protein concentration-dependent changes in reactivity; c, Representative examples of slow protection at 40 nM SRP19 (0.3–0.8 min–1), including C151 (closed circles) and A201
(closed squares); d, Three positions protected rapidly in a protein concentration-independent manner (∼2 min–1), including G153 (closed circles) and
A157 (closed squares); and e, Many nucleotides completely protected by the first time point (20 s) at 20 nM SRP19, including G156 (open circles) and
C189 (open squares), corresponding to a rate ≥5 min–1. In all panels, experiments performed at 20 and 40 nM SRP19 are shown with open and closed
symbols, respectively. The x-axis scales for (b) and (c) differ from (d) and (e). Reactivity is plotted as the ratio of total band intensity normalized to the
intensity in the absence of SRP19 (I / It = 0). Error bars reflecting the 10 s incubation period with iodine are smaller than the symbols used. f, Summary
of static and time-resolved footprinting of phosphorothioate-substituted RNA. Positions protected in both wild type and mutant (A149U) RNAs upon
SRP19 binding are indicated as heavy boxes. Thin boxes denote additional protections observed only with the wild type LS RNA. Positions exhibiting
protein concentration-dependent protection are shown as open circles, whereas the majority were protected at rates independent of protein concentration (closed circles). Some positions showed too small a change in reactivity to be quantified accurately in the time-resolved experiment.
518
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a
b
Fig. 5 Induced fit and cooperativity in early assembly of the human SRP. SRP19 is shown approximately to emphasize that little information exsists
regarding the structure of this protein. a, SRP19 forms a native complex with the LS RNA by induced fit involving at least two intermediates. A rapid
encounter complex forms followed by two steps involving consolidation of the RNA–protein interface. Regions protected in the phosphorothioate
footprinting experiments are emphasized in red (very fast, ≥5 min–1), blue (fast, 1.4 min–1 at 20 nM SRP19) and green (slow, 0.3–0.8 min–1). The position of SRP54 binding is inferred from the homologous Ffh structure23,27. SRP54 may also bind independently to the RNA, but this interaction is probably weak13–16. b, Cooperativity in a three-component ribonucleoprotein mediated by mutual recognition of compatible RNA structures. SRP54 and
the SRP19–domain III RNA structural units both interact at the domain IV helix, but primarily on opposite faces. Backbone positions protected by
binding of the SRP54 homolog, Ffh27, are shown as a green ribbon, and backbone positions protected from hydroxyl radical cleavage upon SRP19
binding are shown in blue; a section protected by both proteins (residues 187–190) is shown in blue. Adenine N1 positions protected from or still
accessible to DMS methylation in the SRP19–RNA complex are shown as red and green spheres, respectively.
Induced fit by architectural RNA binding proteins
Our investigation supports a model for assembly of the
SRP19–RNA ribonucleoprotein by induced fit that accounts for
slow overall complex formation and the requirement for obligatory assembly intermediates (Fig. 5a). SRP19 binds rapidly to the
LS RNA to form an encounter complex. Subsequently, this labile
RNA–protein complex forms a stable, intermediate complex.
Finally, slow consolidation of the RNA–protein interface yields
the native complex. Superposition of both the hydroxyl radical
(Fig. 3c) and phosphorothioate (Fig. 4f) footprints on models of
helices III and IV emphasizes that the RNA structures involved in
assembly of the ribonucleoprotein are localized on one face of
each helix (Fig. 5a). As described below, support for each intermediate or assembly transition was obtained in at least two kinds
of experiments.
unreactive during structural maturation to the native complex, the
encounter complex is a productive intermediate for SRP19–RNA
assembly. Not only does an encounter complex form between a
polyanionic RNA and the highly basic SRP19 protein, but this
encounter complex is structurally specific. The encounter complex
appears to function as a first step in drawing together RNA structural elements into a native RNA–protein complex.
Stable complex
The stable complex (Fig. 5a) is detected by nitrocellulose filter
partitioning (Fig. 2b). This same complex at the same assembly
rates is also detected in independent experiments as proteindependent protection of residues A149 and A199 (Fig. 4f; compare Figs 2a and 4b). Although rearrangement from the
encounter to the stable complex is formally a unimolecular step,
we infer that this complex forms in a protein concentrationEncounter complex
dependent manner because the encounter complex is in rapid
Direct evidence for the encounter complex (kobs ≥ 5 min–1) comes preequilibrium with free RNA and SRP19. This preequilibrium
from the observation that 14 nucleotides in domains III and IV are preceding stable complex formation also accounts, in part, for an
completely protected from cleavage by the first time point at 20 s apparent kon that is much slower than diffusion-limited.
(Fig. 5a, red regions). This state is kinetically labile because stable
complex formation, monitored by nitrocellulose partitioning, Native complex
occurs significantly more slowly at these protein concentrations Almost half of the RNA positions footprint at a concentration(Fig. 2b). Formation of an encounter complex is supported in inde- independent rate much slower than observed for formation of
pendent experiments as reproducible footprinting of the A149U the stable complex (Fig. 5a, green positions). It is this
mutant. No complex is observed with the mutant RNA by filter stable→native transition that accounts for the orders-of-magnibinding (Fig. 2d); however, SRP19 binding to the A149U mutant tude disagreement between koff / kon and the equilibrium Kd.
protects exactly those positions protected in the first 20 seconds of Time resolved footprinting also reports this transition in a third
assembly (Fig. 4f). Since these rapidly protected positions remain way as the lag observed at A205 and G206 (Fig. 4b). Only after
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this slow consolidation of structure is complete does the RNA (2-ME). Denaturing reactions contained 50%(v/v) formamide. Sites
by primer extension33,34. In
show the global pattern of protection characteristic of the native of DMS modification were identified
35,36, 5′-32P-end labeled LS RNA
hydroxyl
radical
cleavage
reactions
structure (Figs 3c, 4f).
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was incubated with SRP19 in the absence of triton and BSA.
Structural origin of cooperativity
The bacterial SRP RNA and domain IV in the human SRP RNA,
and the Ffh and SRP54 proteins are structurally homologous23,26.
Prior work23,27 on the Ffh–bacterial RNA complex therefore provides a framework for understanding how SRP19 stabilizes association of SRP54 with the SRP RNA. Backbone interactions
identified between Ffh and the bacterial RNA27 are superimposed on a helix IV model as a green backbone (Fig. 5b) and
emphasize that Ffh/SRP54 interacts at one face of the helix. The
adenosine N1 positions protected from DMS upon SRP19 binding are shown in red, and the phosphoribose backbone protected
from hydroxyl radical cleavage is shown in blue. The interface
with SRP19 and domain III spans one side of one and a half helical turns of helix IV from the asymmetric bulge to the apical
loop. Contacts are made to exactly the same region as bound by
Ffh/SRP54 but on approximately the opposite side of the domain
IV helix (Fig. 5b). SRP54 and SRP19 or other regions in the RNA
may also interact directly. However, this work supports a model
in which mutual recognition of compatible RNA structures plays
a significant role in SRP19–SRP54 cooperativity.
Iodine cleavage of phosphorothioate-substituted RNA. 8 nM
5′-32P-end labeled, phosphorothioate substituted24,25 RNA was incubated with either 20 or 40 nM SRP19. Assembly was initiated by addition of RNA to a small volume containing SRP19. Aliquots were
removed and cleavage was initiated by addition of 1/10 volume 1 mM
I2 in ethanol (25 °C, 10 s) and quenched by addition of 1/10 volume
10 mM 2-ME and an equal volume of stop dye. Iodine and quench
solutions were stored on ice until use. Band intensities at each time
point were normalized to the intensity observed in the absence of
SRP19. Experiments were repeated for each nucleotide 2–3× at each
protein concentration; standard deviations were ±30% or less. Rates
were obtained by fitting to: I / It = 0 = Ae–kt + B.
Modeling. Structural models of domain III (ref. 37) and domain IV
(ref.23) were constructed for the human RNA using Sybyl (Tripos,
Inc.); steepest descent minimization was used to regularize RNA
geometry.
Acknowledgments
This work was supported by grants from the Searle Scholars Program of the
Chicago Community Trust and by the NIH to K.M.W. We are indebted to K. Henry
and H. Fried for gifts of plasmids and many helpful interactions in early phases of
this work. We thank P. Bevilacqua and E. Westhof for helpful discussions;
M. Been and T. Hall for careful readings of the manuscript; and L. Pedersen and
L. Perera for assistance with RNA modeling.
Signature for assembly intermediates
Many proteins function in an architectural way to stabilize an
active RNA state, obligatorily involving induced fit in the RNA.
Disagreement between the ratio of the dissociation and associa- Correspondence should be addressed to K.M.W. email: [email protected]
tion rate constants, determined in a partitioning experiment, Received 11 December, 2000; accepted 27 March, 2001.
and the equilibrium binding constant provides evidence for an 1. Draper, D.E. Annu. Rev. Biochem. 64, 593–620 (1995).
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native, protein–RNA intermediate complex has formed (Fig. 5a). 3.
4. Walter, P. & Blobel, G. Cell 34, 525–533 (1983).
By this criteria, there is evidence that stable intermediates occur 5. Gundelfinger, E.D., Krause, E., Melli, M. & Dobberstein, B. Nucleic Acids Res. 11,
(1983).
in other ribonucleoprotein assembly reactions28–30. Key features 6. 7363–7374
Lutcke, H. et al. J. Cell Biol. 121, 977–985 (1993).
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Methods
General. SRP19 was overexpressed in E. coli JM109(DE3)pLysS cells
and purified using a modification of the approach described in
Henry et al.11 that yielded nucleic acid-free protein. LS RNA was
transcribed from plasmid phR10, purified by denaturing electrophoresis and refolded31. Reactions were performed at 25 °C in
20 mM Hepes (pH 7.6), 300 mM potassium acetate, pH 7.6, 5 mM
MgCl2, 0.01% (v/v) triton X-100, supplemented with one-fifth volume of SRP19 in protein dilution buffer (50 mM sodium phosphate,
pH 8, 300 mM NaCl and 0.5 mg ml–1 bovine serum albumin).
Stoichiometric binding experiments demonstrated that at least 90%
of the RNA and protein components were competent for assembly,
assuming a 1:1 complex.
Binding and kinetic experiments. Equilibrium RNA–protein
binding reactions were filtered through nitrocellulose and Hybond
(Amersham) membranes using a dot blot apparatus32. To determine
kon, aliquots were quenched using 1,000-fold excess unlabeled LS
RNA and immediately filtered on the double filter system. The koff
was measured by preforming an RNA–SRP19 complex and diluting
the complex 200-fold or by addition of 5,000-fold excess unlabeled
competitor LS RNA prior to filtering.
Footprinting. DMS was added to reactions containing RNA or
RNA–protein complexes19 and quenched with 2-mercaptoethanol
520
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