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INTRODUCTION |
The targeting of nascent secretory and integral membrane proteins
to the endoplasmic reticulum of eukaryotes and the inner membrane of bacteria has been extensively studied in recent years, and
many details of the pathway have been uncovered (1, 2). In eukaryotes,
targeting begins when the N-terminal signal sequence of a nascent
secretory protein being synthesized by the ribosome is specifically
recognized by the SRP541
subunit of the signal recognition particle. SRP bound to the ribosome-nascent chain complex interacts with its receptor in the
endoplasmic reticulum membrane, and subsequently the ribosome-nascent chain complex is passed to the translocon. A tight junction formed between the ribosome and the translocon then allows cotranslational vectorial transfer of the nascent polypeptide chain into the
endoplasmic reticulum lumen.
In contrast, prokaryotic cells use multiple pathways to target nascent
protein chains to their final destinations (3). The SRP
homologue in Escherichia coli is part of a
specialized system responsible for bringing polytopic inner membrane
proteins to the cell membrane for cotranslational insertion (4-8).
Compared with the eukaryotic SRP, which contains six protein subunits
bound to a 7SL RNA scaffold, the E. coli SRP consists
of a single protein subunit (Ffh, for
fifty-four homologue) that is bound
to a smaller but homologous RNA termed 4.5S RNA (1). Ffh and SRP54
share similar two-domain modular structures with an N-terminal GTPase (NG) domain and a C-terminal methionine-rich M domain (9, 10) that
binds to both RNA and signal sequences (11-13). The relative simplicity of the E. coli SRP makes it a tractable model for
the eukaryotic system.
Signal sequences function as molecular zip codes, specifying the
destination of each nascent chain. Despite a lack of consensus among
the signal sequences of different proteins, they do share some very
general characteristics, such as an N-terminal region that is typically
positively charged (the n-region) followed by a hydrophobic core of
7-13 amino acids (the h-region) and a C-terminal polar sequence
containing a cleavage site recognized by signal peptidase (the
c-region) (14). How such divergent sequences are specifically
recognized by SRP has remained a puzzle for many years. High resolution
structures of the M domains of Ffh and SRP54 suggest that signal
sequences may be bound by a hydrophobic "finger" loop that has
sufficient flexibility to mold itself to a wide variety of sequences
(15, 16). Based on a crystal structure of the Ffh M domain complexed
with 4.5S RNA, Batey et al. (17) recently proposed that an
RNA surface contiguous with the finger loop cleft might contribute to
the recognition of the positively charged n-region of the signal
sequence. Whether these proposed models faithfully represent signal
sequence recognition will have to await a structure of SRP54 or Ffh in
a complex with a signal peptide.
The structural and functional consequences of signal sequence binding
to SRP have long been the subject of intensive investigation. One
attractive hypothesis that has been tested by a number of laboratories
is that signal sequence binding to SRP54 or Ffh regulates the binding
and/or hydrolysis of nucleotides by the NG domain. In related studies,
isolated peptides corresponding to the signal sequence of the E. coli outer membrane protein, LamB, were shown to inhibit the
GTPase activity of the E. coli and canine pancreas SRPs by
blocking nucleotide binding (18, 19). Conversely, however, Bacher
et al. (20) demonstrated that signal sequences presented to
SRP as ribosome-nascent chain complexes increased GTP binding to SRP.
These data were reconciled with previous results by proposing that a
ribosomal component overcomes the signal sequence-induced inhibition of
nucleotide binding. Finally, an elegant study by Rapiejko and Gilmore
(21) clearly shows that the nucleotide-binding site in SRP54 can remain
empty until its high affinity interaction with SRP receptor, arguing
that signal sequence binding to SRP54 has little or no effect on GTP
binding. In the arena of structural effects, peptides corresponding to
functional signal sequences have been shown to destabilize the tertiary
structure of isolated Ffh, an effect that can be reversed by the
addition of 4.5S RNA (22). These studies would seem to indicate that
signal sequences inhibit, enhance, or have no effect on nucleotide
binding to SRP depending upon the experimental conditions used. Here we
present an explanation for this apparent contradiction.
Our work demonstrates that experiments conducted with isolated peptides
corresponding to signal sequences must be carried out and interpreted
with extreme caution. We initially observed aggregation of SRP by
signal peptides when making concentrated samples of Ffh·4.5S
RNA plus LamB signal peptide analogues for structural studies by
NMR. In an attempt to find conditions to circumvent this aggregation,
we discovered that signal peptide-induced aggregation of SRP occurs
even at much lower concentrations, such as those used to show that
signal peptides inhibit the SRP GTPase activity (18, 19). In this
study, we present evidence that peptides with the features of signal
sequences (e.g. a positively charged region followed by a
region of substantial hydrophobicity) nonspecifically bind and
aggregate RNA molecules at micromolar concentrations. In the case of
4.5S RNA complexed with Ffh, signal peptide-induced RNA aggregation
results in the aggregation of the entire SRP particle, which is the
likely cause for its loss of GTPase activity.
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EXPERIMENTAL PROCEDURES |
Preparation of Proteins, Peptides, and RNA--
Ffh and 4.5S RNA
were prepared as described previously (22). 5S rRNA and lysine-specific
tRNA were purchased from Roche Molecular Biochemicals and Sigma,
respectively. 7SL RNA from canine pancreas was a generous gift from
Harris Bernstein (National Institutes of Health). KRR-LamB, DM,
R1, OmpA, and K5W(AL)10 peptides were synthesized and purified according to previously published methods (23). The purity and correct mass of each peptide were verified by high
pressure liquid chromatography and mass spectrometry, and the
concentration of each was determined by quantitative amino acid analysis.
GTPase Activity Assays--
GTPase assays were conducted as
described previously (19), with the exception that the concentrations
of Ffh protein and 4.5S RNA were 25 and 50 nM,
respectively. Reactions in buffer A (50 mM
triethanolamine/acetic acid, pH 7.5, 25 mM potassium acetate (K acetate), 2.5 mM magnesium acetate, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM
dithiothreitol, 0.1% octaethylene glycol monododecyl ether (19)) were
allowed to continue for 20 min before quenching. However, because of
the low basal GTPase rate of Ffh at higher salt concentrations, GTPase
activity assays in buffer A supplemented with K acetate (175 mM final concentration) were run for a full hour to
generate adequate signal.
Aggregation Tests--
To test for aggregation of Ffh under the
conditions of the GTPase activity assay, 0.5-ml samples of Ffh ± 4.5S RNA were prepared with the indicated concentrations of signal
peptides exactly as indicated for the GTPase inhibition assay. After
the addition of "cold" GTP and 30 min of incubation at room
temperature, samples were centrifuged for 15 min in a microcentrifuge.
Supernatants were carefully removed from the resultant pellets and
precipitated by the addition of 0.5 ml of 20% trichloroacetic acid,
incubated for 20 min on ice, and centrifuged for 15 min as before.
Pellets and trichloroacetic acid precipitates were resuspended in
SDS-polyacrylamide gel sample buffer, run on 10% SDS-polyacrylamide
gels, and visualized by Coomassie Blue staining. To test for
aggregation of RNAs, each RNA (200 nM, except where
indicated otherwise) was incubated with the indicated concentrations of
signal peptides in GTPase assay buffer A at room temperature for 30 min. UV absorption spectra (230-300 nm) of these solutions were
recorded, samples were microcentrifuged for 15 min, and the UV
absorbance of the supernatants was remeasured for comparison.
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RESULTS |
A LamB Signal Peptide Analogue That Inhibits the Ffh·4.5S RNA
GTPase Activity Also Aggregates the Complex in the Same Concentration
Range--
Miller et al. (19) demonstrates that a
peptide corresponding to the wild-type LamB signal sequence (Table
I) causes dose-dependent inhibition of the Ffh·4.5S RNA GTPase activity. A LamB signal peptide
analogue containing an insertion of three basic residues in the
n-region termed KRR-LamB (Table I) behaves similarly (22). This
insertion increases the aqueous solubility of the LamB signal peptide
(24) but is not expected to affect its in vivo function because the insertion of two basic residues in the n-region of the LamB
signal sequence does not affect its ability to target nascent LamB
protein (25). KRR-LamB inhibited the GTPase activity of the Ffh·4.5S
RNA complex with an IC50 of 9.4 µM and with
significant positive cooperativity (Hill coefficient = 5, Fig.
1a). Identical samples were
then tested for signal peptide-induced Ffh·4.5S RNA aggregation by
brief centrifugation as described under "Experimental Procedures."
Ffh partitioned into the pellet fraction upon the addition of KRR-LamB
with roughly half of the Ffh located in the pellet at a KRR-LamB
concentration of 3-6 µM (Fig. 1b). This
aggregation had previously escaped detection, presumably because it was
not accompanied by any obvious change in sample turbidity, indicating that the aggregates were fairly small. These results clearly indicate that KRR-LamB aggregates Ffh·4.5S RNA in the same concentration range
in which inhibition of the Ffh·4.5S RNA GTPase activity occurs.

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Fig. 1.
The KRR-LamB signal peptide aggregates the
Ffh·4.5S RNA complex and inhibits its GTPase activity in the same
concentration range. a, the Ffh·4.5S RNA GTPase
activity was determined (as described under "Experimental
Procedures") at the indicated concentrations of KRR-LamB signal
peptide and compared with a control reaction without added peptide. The
curve reflects a nonlinear fit to the data with
IC50 = 9.4 ± 0.9 µM and
nH = 5, indicating significant positive
cooperativity. Ffh·4.5S RNA (b) or Ffh alone
(c) were tested for KRR-LamB-induced aggregation by brief
centrifugation as described under "Experimental Procedures." The
first two lanes of each gel show the Ffh band from the
trichloroacetic acid precipitates (lane 1) and pellets
(lane 2) from a sample of Ffh·4.5S RNA (b) or
of Ffh alone (c) in the absence of added peptide.
Lanes 3-9 of each gel show the Ffh band from pellets after
incubation with 1.2, 3, 6, 12, 30, 60, and 120 µM
KRR-LamB, respectively. Molecular mass markers are indicated to
the left of each gel in kDa.
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Aggregation of Ffh·4.5S RNA by KRR-LamB Is Mediated by a Direct
Interaction with 4.5S RNA--
Surprisingly, in similar aggregation
tests conducted on Ffh alone in the absence of 4.5S RNA, Ffh did not
pellet even at the highest concentration of KRR-LamB tested (120 µM, Fig. 1c), demonstrating that the RNA
component must be present for aggregation to occur. To determine
whether this is a direct effect of KRR-LamB on 4.5S RNA, we incubated
4.5S RNA alone and with increasing concentrations of KRR-LamB and then
compared the UV absorbance spectra of these samples before and after
brief centrifugation. Surprisingly, the addition of KRR-LamB to 4.5S
RNA resulted in a dose-dependent loss of RNA from solution
after centrifugation (Fig. 2,
a and b). Half-maximal aggregation of 4.5S RNA by
KRR-LamB occurred at a peptide concentration of 6 µM,
which was directly comparable to the concentration of KRR-LamB that
caused 50% aggregation and inhibition of the GTPase activity of the
Ffh·4.5S RNA complex. Interestingly, when KRR-LamB was added to 4.5S
RNA, the RNA absorbance increased in a dose-dependent
manner with an EC50 of 6-12 µM (Fig. 2,
a and c). This increase was not attributable to
absorbance of KRR-LamB itself because this peptide had no aromatic
residues and did not absorb in this region (Fig. 2a,
inset).

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Fig. 2.
The aggregation of Ffh·4.5S RNA
by KRR-LamB is mediated by a direct interaction with 4.5S RNA.
a, 4.5S RNA was tested for KRR-LamB-induced aggregation by
brief centrifugation after incubating 4.5S RNA with various
concentrations of peptide as described under "Experimental
Procedures." Solid curves with filled symbols
and dashed curves with open symbols indicate
absorbance spectra recorded before and after centrifugation,
respectively. Circles, no KRR-LamB added; inverted
triangles, 1.2 µM KRR-LamB; diamonds, 6 µM KRR-LamB; triangles, 12 µM
KRR-LamB; squares, 30 µM KRR-LamB. The
inset at bottom is the absorption spectrum of 6 µM KRR-LamB alone. These data are represented in graphic
form in panel b in which the loss of RNA from solution with
increasing KRR-LamB concentration is indicated by the ratio of
absorbance at 260 nm after centrifugation to the absorbance before
centrifugation. Panel c graphically represents the
increase in RNA absorbance that occurs with KRR-LamB addition. The
percent increase in 260 nm absorbance at each peptide concentration was
calculated relative to the absorbance of 4.5S RNA alone.
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KRR-LamB Aggregates RNA Nonspecifically--
To determine whether
KRR-LamB aggregates 4.5S RNA via a specific binding interaction,
several different RNA species were tested for aggregation by this
peptide including 7SL RNA from dog pancreas (the mammalian SRP RNA), 5S
rRNA from E. coli, and lysine-specific tRNA from E. coli (Fig. 3). Each RNA alone did
not pellet when centrifuged (black bars); however,
incubation of each RNA with 6 µM KRR-LamB resulted in
significant aggregation (dark gray bars). All of the
heterologous RNAs were even more sensitive to aggregation by KRR-LamB
than was 4.5S RNA. In each case, the aggregation was accompanied
by a significant increase in the 260 nm absorbance of each RNA (data
not shown) as was observed with 4.5S RNA. These results show that
KRR-LamB aggregates RNA nonspecifically.

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Fig. 3.
The interaction between signal peptides and
RNA is nonspecific. 7SL RNA, 5S rRNA, tRNA, and 4.5S RNA
were incubated at 200 nM either alone (black
bars) or with 6 µM KRR-LamB peptide (dark gray
bars). 4.5S RNA at 300 nM was incubated with 5 µM OmpA signal peptide (light gray bar) and 2 µM K5W(AL)10 (white
bar). The fraction of RNA remaining in solution was
calculated from the ratio of the 260 nm absorbance after centrifugation
to the absorbance before centrifugation.
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Other Signal Peptides Also Aggregate RNA--
A peptide
corresponding to the signal sequence of the E. coli outer
membrane porin, OmpA, as well as an idealized peptide with a longer
hydrophobic core (K5W(AL)10, see Table I) were tested for aggregation of 4.5S RNA. As seen in Fig. 3, incubation with
5 µM OmpA signal peptide pellets 90% of the total 4.5S
RNA, and the addition of 2 µM
K5W(AL)10 pellets 71% of the total 4.5S RNA.
These results indicate that this phenomenon is not limited to LamB and
may be characteristic of signal peptides in general.
The Hydrophobic Core Plays an Important Role in Aggregation of the
Ffh·4.5S RNA Complex by Peptides--
To understand what
characteristics of signal peptides are important for aggregation of the
Ffh·4.5S RNA complex, we tested peptides corresponding to functional
and nonfunctional analogues of the LamB signal sequence. The DM
deletion mutant peptide is missing four residues from the hydrophobic
core (Table I), leading to a defect in translocation of LamB nascent
chains in vivo (26); the lower helical propensity of this
peptide compared with wild type has been proposed to be responsible for
its in vivo defect (27). When added to GTPase reactions, DM
does not inhibit the Ffh·4.5S RNA GTPase activity even at the highest
concentration tested (30 µM, Fig.
4a) as shown earlier (19).
Interestingly, DM also did not aggregate the Ffh·4.5S RNA complex.
Even in the presence of 30 µM DM peptide, no Ffh was
pelleted by centrifugation (Fig. 4b). Similar results were
obtained with a LamB variant containing an aspartate substitution
within the hydrophobic core (data not shown), confirming that the
signal peptide hydrophobic core plays an important role in the
aggregation of RNA.

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Fig. 4.
Nonfunctional signal peptides do not
aggregate Ffh·4.5S RNA or inhibit its GTPase activity.
a, the Ffh·4.5S RNA GTPase activity was measured with the
indicated concentrations of DM ( ) and R1 ( ) peptides and compared
with control reactions without added peptides. The curve
indicates the best nonlinear fit to the R1 data with IC50 = 0.94 ± 0.05 µM and nH = 1.5 ± 0.1. The Ffh·4.5S RNA complex was tested for aggregation by DM
(b) or R1 (c) by brief centrifugation. The
first two lanes of each gel show the Ffh band from the
trichloroacetic acid precipitates (lane 1) and pellets
(lane 2) from a sample of Ffh·4.5S RNA in the absence of
added peptide. In b, lanes 3-10 show the Ffh
band from pellets after incubation with 0.2, 0.3, 0.6, 1.5, 3.1, 6.1, 15, and 31 µM DM peptide. Lanes 3-9 of
panel c show the Ffh band from pellets after incubation with
0.04, 0.08, 0.21, 0.42, 0.84, 2.1 and 4.2 µM R1 peptide.
Molecular mass markers are indicated to the left of each gel
in kDa.
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The R1 pseudorevertant peptide lacks four hydrophobic core residues
like DM (Table I) but has higher helical propensity than does DM due to
the replacement of a helix-breaking glycine residue with cysteine (27).
The R1 signal sequence effectively targets LamB nascent protein
in vivo (26). Paralleling what has been shown earlier (19),
R1 inhibited the Ffh·4.5S RNA GTPase activity with an
IC50 of 0.94 µM (Fig. 4a). It is
interesting to note that R1 inhibited the Ffh·4.5S RNA GTPase
activity with significantly less cooperativity than did KRR-LamB with a
Hill coefficient of only 1.5 compared with 5 for KRR-LamB (Fig.
1a). Strikingly, in the same concentration range where R1
inhibited the Ffh·4.5S RNA GTPase activity, R1 also aggregated
Ffh·4.5S RNA (50% aggregation at ~0.84 µM, Fig.
4c). These experiments indicate that both hydrophobicity and
helical propensity are important characteristics of peptides that
aggregate RNA.
Electrostatics Also Make an Important Contribution to the
Aggregation of Ffh·4.5S RNA by Signal Peptides--
To investigate
the effect of ionic strength on the peptide/RNA interaction, we
increased the potassium acetate concentration in GTPase assay buffer A
(19) from 25 to 175 mM. Under these more physiological
conditions, the ability of KRR-LamB to aggregate either the Ffh·4.5S
RNA complex or 4.5S RNA alone was reduced approximately 5-fold; 12-30
µM KRR-LamB aggregated 50% of the total Ffh·4.5S RNA
(Fig. 5a) compared with only
3-6 µM KRR-LamB that was required to have the same
effect in low salt (Fig. 1b). Likewise, 30 µM
KRR-LamB aggregated 50% of the total 4.5S RNA alone under these
conditions compared with 6 µM KRR-LamB in low salt buffer
(Fig. 5b, compare with Fig. 2b). It is noteworthy that the half-maximal increase in RNA absorption was also shifted to
higher KRR-LamB concentrations in 175 mM K acetate (30-60
µM as shown in Fig. 5c, compared with 6-12
µM in buffer A as shown in Fig. 2c).

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Fig. 5.
Increasing the salt concentration to a
physiological level reduces the ability of KRR-LamB to aggregate 4.5S
RNA and inhibit the Ffh·4.5S RNA GTPase activity by similar
degrees. a, aggregation of the Ffh·4.5S RNA complex
by KRR-LamB was tested as in Fig. 1b, except that the
concentration of K acetate in the buffer was increased from 25 to 175 mM. Lanes 1 and 2 show the Ffh band
from the trichloroacetic acid precipitate and pellet, respectively,
from a sample of Ffh·4.5S RNA without added peptide. The Ffh band
from pellets after incubation of Ffh·4.5S RNA with 1.2, 3, 6, 12, 30, 60, and 120 µM KRR-LamB are shown in lanes
3-9. Molecular mass markers are indicated to the left
of the gel in kDa. b and c, aggregation of
4.5S RNA by KRR-LamB was tested as in Fig. 2a with the
exception of [K acetate] = 175 mM. Panel b
illustrates the loss of RNA from the solution at higher KRR-LamB
concentration as calculated in Fig. 2b, and panel c shows
the enhancement of RNA absorbance at high KRR-LamB concentration, as
calculated in Fig. 2c. Inhibition of the GTPase activity of Ffh·4.5S
RNA by KRR-LamB at 25 mM K acetate (gray bars)
and 175 mM K acetate (black bars) is shown in
the bar graph in panel d.
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Because the signal sequence/Ffh interaction is thought to be primarily
hydrophobic in nature, we initially hypothesized that increasing the
ionic strength of the buffer would strengthen peptide binding to its
specific site on Ffh, possibly resulting in a lower IC50
for GTPase inhibition. Contrary to this idea, however, the amount of
KRR-LamB required to inhibit 50% of the total Ffh·4.5S RNA GTPase
activity was increased to 60-120 µM in 175 mM K acetate (Fig. 5d) compared with 9.4 µM at low salt (Fig. 1a). When the buffer
ionic strength was increased, the ability of KRR-LamB to both aggregate
4.5S RNA and inhibit the GTPase activity of Ffh·4.5S RNA was reduced
by similar degrees, suggesting that the aggregation and GTPase
inhibition are related events. Intriguingly, however, at low KRR-LamB
concentrations in which significant aggregation did not occur (<30
µM), there appeared to be a subtle increase in Ffh·4.5S
RNA GTPase activity (32% increase over control at 6 µM
KRR-LamB, Fig. 5d). It is tempting to speculate that the increase in ionic strength has reduced the aggregation to an extent where the true effect of signal peptide binding to its site on Ffh is uncovered.
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DISCUSSION |
In this study, we show that signal peptides do not inhibit the
E. coli SRP GTPase activity directly, as originally proposed (19), but instead cause aggregation of the entire SRP particle via a
nonspecific interaction with the RNA component. We propose that the SRP
GTPase activity is lost secondarily because of aggregation. The
"nonfunctional" signal peptides used as negative controls in these
earlier studies (18, 19) are unable to aggregate RNA, which explains
their lack of effect on the SRP GTPase activity. In the simplest
interpretation, the Ffh protein plays a passive role and is present in
aggregates only because it exists in a high affinity complex with 4.5S
RNA. These data suggest that the same signal sequence characteristics
that are important for SRP recognition, i.e. an N-terminal
charged region, a hydrophobic core, and helical propensity, also
promote efficient but nonspecific RNA aggregation. It is important to
point out that we have no evidence for an in vivo
interaction between signal sequences and RNA. Instead, we believe this
to be purely an artifact of in vitro experiments that
use high concentrations of signal peptides under conditions of
low ionic strength. Inside the cells, signal sequences are
presumably prevented from aggregating RNA because they are tethered to
long nascent chains and/or ribosomes and are presented at physiological
concentrations and physiological ionic strength. It is an intriguing
possibility that signal sequences are further isolated from RNA by
binding to chaperone or targeting proteins. Nevertheless, under these
well controlled cellular conditions, the n-region of signal sequences
in fact may productively bind to the SRP RNA as proposed previously
(17).
The mechanism for this aggregation, although not central to the issue
at hand, still remains a puzzle. Any model for this peptide-induced RNA
aggregation must take into account the electrostatic, hydrophobic, and
structural elements of the interaction. Because increasing the salt
concentration to a physiological level reduces peptide-induced
aggregation of the RNA (Fig. 5b), we propose that positively
charged side chains in the n-region of the signal peptide interact with
the negatively charged phosphodiester backbone of the RNA. The
hydrophobic core of the signal peptide also plays an important role as
illustrated by the result that the deletion of four hydrophobic
residues (DM peptide) or the insertion of a negative charge into the
hydrophobic core severely diminishes Ffh·4.5S RNA aggregation (Fig.
4b). Furthermore, aggregation is restored in the R1
pseudorevertant peptide, which has similar hydrophobicity to DM but a
higher propensity to form a helix (Fig. 4c). This result
implicates peptide helical propensity in RNA aggregation.
Finally, functional signal peptides cause a dose-dependent increase in RNA UV absorption (Figs. 2c and 5c).
We propose the induced hyperchromicity to be a result of base
unstacking (28), although the possibility of light scattering has not
been eliminated.
Some of the characteristics of this interaction are reminiscent of the
aggregation of DNA and RNA by polybasic sequences such as spermine and
protamines (28, 29). Protamines are small arginine-rich proteins that
function to tightly package DNA in sperm cells by neutralizing the
charge of the DNA backbone using short sequences of 4-6 arginine
residues. Multiple DNA molecules can be tethered together by binding
different arginine stretches of the same protamine molecule (30). The
molecular details of the binding are a topic of some dispute; however,
a Raman spectroscopy study suggests that protamine binding to DNA is
accompanied by significant base unstacking (31). Furthermore, the
binding of protamines to DNA is nonspecific, strongly cooperative,
causes DNA aggregation, and can be inhibited by increasing the buffer ionic strength (32). Protamines have also been shown to bind RNA
nonspecifically, and they can aggregate and precipitate tRNA (33). The
peptides used in the current study each contain a single stretch of
basic amino acids, 2-5 residues in length, which we propose interacts
with the RNA backbone and may cause significant base unstacking as is
observed for protamine/DNA interactions. We speculate that this
interaction shields the RNA backbone charges and also results in the
coating of the RNA with the hydrophobic cores of the peptides. In our
view, these two effects combined could then lead to association of the
RNA with other shielded and coated RNAs, yielding small
aggregates. Whether this model adequately represents the true
nature of the physical interaction between the RNA and peptides studied
here remains an open question. On the other hand, for our interests
here, how signal peptides aggregate RNA is not the central issue. What
is important is that they do aggregate RNA in vitro and that
the discovery of this artifact forces us to rethink the generally
accepted scenario of events that occurs when nascent polypeptides
are targeted by SRP.
For all peptides and conditions tested, the ability of a peptide to
inhibit the Ffh·4.5S RNA GTPase activity was mirrored by its efficacy
for aggregating the RNA component of that complex. Therefore, we
conclude that the inhibition of the Ffh·4.5S RNA GTPase activity by
signal peptides is a direct result of aggregation, leading to a loss of
Ffh GTPase function, and not the result of allosteric modulation of the
GTPase site upon signal peptide binding to Ffh as proposed previously
(19). Furthermore, because functional signal peptides also aggregate
7SL RNA (Fig. 3), we extend these conclusions to the in
vitro study of signal peptide interactions with mammalian SRP as
well (18). These two studies of Miller et al. (18, 19) have
been interpreted as indicating that signal sequence binding to SRP
causes the release of nucleotide, stabilizing an empty site form. Our
results suggest that this interpretation is unwarranted and that any
real effect of signal peptides on SRP is obscured by an artifactual
interaction with the SRP RNA. In fact, the increase in GTPase activity
at low KRR-LamB concentrations under conditions in which aggregation is
reduced (Fig. 5d) provides a tantalizing hint that signal
peptides may enhance the GTPase activity. A recent study by Zheng and
Nicchitta (34) demonstrates that eukaryotic SRP has limited affinity
for the wild-type LamB sequence, but a variant in which several
hydrophobic core residues are mutated to leucine is efficiently
targeted. Perhaps choosing a signal peptide sequence with higher
affinity for Ffh or SRP54 combined with careful control of aggregation
will finally allow determination of the true effect, if any, of signal
sequence binding on the nucleotide-binding domain.
If signal peptides inhibit the Ffh·4.5S RNA GTPase activity
artifactually, by what mechanism do they inhibit the GTPase activity of
the isolated Ffh protein as reported previously (22)? The interaction
between LamB signal peptides and RNA is only one example of the unusual
chaotropic behavior these peptides can exhibit. It has been
demonstrated that the binding of functional signal peptides to the
isolated Ffh protein destabilizes the Ffh tertiary structure as
observed by limited proteolysis (22). In our current thinking,
inhibition of the Ffh GTPase activity by functional signal peptides is
just a symptom of this overall loss of structural integrity.
This work demonstrates the extreme care that must be taken in
conducting and interpreting experiments with isolated signal peptides.
Signal peptides are known to spontaneously insert into membranes
(35-39) and have been shown here to bind and aggregate RNA
nonspecifically; they are also prone to self-association at high
concentrations. These propensities must carefully be considered in the
experimental design of in vitro experiments involving
peptides with the characteristics of signal sequences.