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J. Biol. Chem., Vol. 277, Issue 43, 41157-41162, October 25, 2002
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§¶,
,, and**
From the Institute of Molecular Biology, Academia
Sinica, Taipei 115, Taiwan, the § Institute of Molecular
Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan, and the Institute of Biochemistry, National
Yang-Ming University, Taipei 112, Taiwan
Received for publication, July 3, 2002, and in revised form, August 12, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The Escherichia coli RNA degradosome
is a multicomponent ribonucleolytic complex consisting of three major
proteins that assemble on a scaffold provided by the C-terminal region
of the endonuclease, RNase E. Using an E. coli two-hybrid
system, together with BIAcore apparatus, we investigated the ability of
three proteins, polynucleotide phosphorylase (PNPase), RhlB RNA
helicase, and enolase, a glycolytic protein, to interact physically and
functionally independently of RNase E. Here we report that Rh1B can
physically bind to PNPase, both in vitro and in
vivo, and can also form homodimers with itself. However, binding
of RhlB or PNPase to enolase was not detected under the same
conditions. BIAcore analysis revealed real-time, direct binding for
bimolecular interactions between Rh1B units and for the RhlB
interaction with PNPase. Furthermore, in the absence of RNase E,
purified RhlB can carry out ATP-dependent unwinding of
double-stranded RNA and consequently modulate degradation of
double-stranded RNA together with the exonuclease activity of PNPase.
These results provide evidence for the first time that both functional
and physical interactions of individual degradosome protein components
can occur in the absence of RNase E and raise the prospect that the
RNase E-independent complexes of RhlB RNA helicase and PNPase,
detected in vivo, may constitute mini-machines that assist
in the degradation of duplex RNA in structures physically distinct from
multicomponent RNA degradosomes.
RNA metabolism is a complex process affecting the control of gene
expression. In bacteria, a multicomponent ribonucleolytic complex
termed the RNA degradosome (1-4) has been identified as playing an
important role in the control of mRNA degradation (for recent
reviews, see Refs. 5-11). The multicomponent complex consists of: the
RNA endonuclease RNase E, whose activity is essential for
Escherichia coli cell growth (12-14), RNA processing (15, 16), and degradation (17, 18); the 3'-5' exoribonuclease PNPase
(19); RhlB RNA helicase (20); and enolase (21), an enzyme involved in
the glycolytic pathway and other chaperonin proteins (3, 22).
Interestingly, in addition to mRNAs, highly structured, stable RNA
fragments have also been found to be associated with RNA degradosome
complexes (3, 23), which implies quality control by the RNA degradosome
for the biogenesis of stable RNAs. Degradosome complexity and its
cooperation with individual protein components acting on
degradation-targeted RNA, in vivo, remains to be discovered.
Various approaches revealing protein-protein interactions in the
degradosome indicate that the C-terminal region of RNase E serves as a
scaffold that directly binds PNPase, RhlB RNA helicase, and enolase
(24, 25). No other interactions among these component proteins have
been detected (25) or reported. Recently, a mini-degradosome complex
(26) containing the scaffold region (without the N-terminal enzymatic
region of RNase E), RhlB RNA helicase, and PNPase was reconstituted
in vitro. These experiments revealed a functional interaction between RhlB RNA helicase and PNPase: RhlB helicase bound
to the RNase E C-terminal region leads to subsequent ATP-activated degradation of a stem-loop segment intermediate RNA by PNPase. In
the absence of the RNase E C-terminal region, RNA degradation does not
occur. This led to the proposal that interaction between both RNase
E-bound PNPase and RhlB helicase attacks the 3'-end of a structured
mRNA (26). Recently, it has been shown that, in vivo,
the steady state levels of individual component degradosome proteins
differ and that degradosome protein components can exist both bound and
unbound to RNase E (27). Thus the potential exists for the formation of
protein complexes other than those formed on the RNase E scaffold to
target RNAs for degradation. Here, we have addressed this question by
investigating possible interactions among degradosome protein
components independently of RNase E. We show, both in vivo
and in vitro, that RhlB RNA helicase binds directly to
PNPase and that the binding aids double-stranded RNA degradation by
PNPase. These findings indicate that functionally important
interactions between degradosome components can occur in the absence of
the degradosome scaffold and suggest the existence of a dynamic network
of degradosome components in vivo that targets various kinds
of structured RNAs for rapid degradation by PNPase-RhlB complexes.
Bacterial Strains and Plasmids--
The E. coli
strains BZ99 (his E. coli Two-hybrid Assay--
E. coli two-hybrid
assays were performed on MacConkey agar plates containing 1% maltose
as described (29). Antibiotic concentrations were 100 µg/ml
ampicillin and 34 µg/ml chloramphenicol. The plates were incubated at
30 °C for ~2-3 days; photographs of individual plates were taken
using the Kodak DC120 digital camera (Eastman Kodak) with KDS1D program
(Eastman Kodak).
The strength of interactions between individual pairs of proteins was
determined by assaying Protein Purifications and Western Blot Analyses--
The
FLAG-tagged PNPase, RhlB RNA helicase, and enolase were purified as
previously described (3) except that cell debris was removed from cell
lysate by centrifugation at 18,000 × g
(versus 10,000 × g) for 15 min. To purify
unbound protein components, the supernatant was precipitated with 60%
(instead of 40%) saturation of ammonium sulfate, incubated on ice for
15-30 min and pelleted down by centrifugation at 12,000 × g for 20 min. The pellet was washed with 60% saturated
ammonium sulfate in buffer A (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) and centrifuged at 12,000 × g for 20 min. The pellet was dissolved in buffer A containing 20% glycerol, adjusted to 12.5 ml, and filtered with a 0.45-µm filter. The protein was then purified on an anti-FLAG M2 affinity gel (Sigma) column according to the manufacturer's instruction by using 1 ml of solution of synthetic FLAG oligopeptide (1 mg/ml) (PAN Laboratory, Stanford University). Prior to elution, 1 ml of 0.1 mg/ml FLAG oligopeptide solution and 2 ml of buffer A containing 20% glycerol were used to
remove/wash weakly interacting proteins. For BIAcore surface plasmon
resonance analysis, the purified proteins were then dialyzed against
0.1 M phosphate buffer, pH 7.0, containing 50 mM NaCl at 4 °C overnight. The enzymatic activities were
monitored as described (see later section on activity assays).
BIAcore Surface Plasmon Resonance Analysis--
Real time
protein-protein interactions were examined on a BIAcore instrument
(BIAcore X). RhlB RNA helicase and bovine serum albumin were
immobilized on different flow cells of a CM5 sensor chip using an
amine-coupling kit (Amersham Biosciences). Briefly, the chip
surface was first activated by injection of 35 µl of 1:1 mixture of
0.4 M
N-ethyl-N'-(dimethylaminopropyl) carbodiimide hydrochloride and 0.1 M N-hydroxysuccinimide.
Native RhlB RNA helicase (70 µl of 35 µg/ml) in 100 mM
phosphate buffer, 50 mM NaCl, pH 7.0, (optimal buffer
components as determined by trial preconcentration experiments) was
immobilized on one flow cell, and an additional flow cell was prepared
as a blank background by immobilization of bovine serum albumin (50 µg/ml) under the same buffer conditions. Remaining activated groups
on each flow cell were blocked by injection of 35 µl of 1 M ethanolamine HCl, pH 8.5. The chip was then washed twice
with 10 µl of 0.1 M NaOH to remove any non-covalently
bound proteins. Finally, the system was primed with the running buffer
(100 mM phosphate buffer, pH 7.0, 50 mM NaCl).
The interaction assays were performed with a constant (10, 20, or 30 µl/min as indicated) flow rate at 25 °C. Individually distinct
concentrations, ranging from 5 to 50 nM (as shown in Fig.
3) of purified native RhlB RNA helicase, PNPase, or enolase were
injected as analytes. The chemical binding groups were regenerated by
sequentially washing the analytes with 10 µl of injection of 0.1 M NaOH, 0.1 M glycine-HCl (pH 3.5) and 10%
ethanol until a background level was attained.
Helicase Activity and PNPase Activity Assays--
RNA
oligonucleotides used for RNA helicase unwinding and the PNPase
degradation assay were double-stranded substrates that resulted from
the annealing of a short single-stranded oligo 5'-AGC GCA GUA CC and
its complementary longer single stranded oligo 5'-ACA GUA UUU
GGU ACU GCG CUC U
(synthesized by Dharmacon Research Inc.). Underlined nucleotides
indicate the complementary region with the short strand RNA oligo. The
RNA sequences of long strand RNA are identical to the 5'-end sequences
of RNAI, the antisense RNA of ColEI-type plasmids (18), and contain an
RNase E cleavage site (33-35). Both strands were 5'-end-labeled with
[ Degradosome Protein Components Are Able to Interact in Vivo
Independently of RNase E--
General methods to identify interacting
proteins or to study protein-protein interactions have been developed
extensively. Among them, the two-hybrid systems currently represent the
most powerful in vivo approaches. Here, we applied the
E. coli two-hybrid approach (29) to study possible
interactions among degradosome protein components. Individual
degradosome component proteins were fused to two complementary
fragments of the catalytic domain of Bordetella pertussis
adenylate cyclase into plasmids pT25- and pT18-based vectors, as
described under "Experimental Procedures." As seen in Fig.
1A, the pink/red phenotypes
displayed on MacConkey/maltose plates demonstrates that RhlB RNA
helicase is able to interact not only with the RE12 fragment (residues
684-784 of RNase E region are sufficient for interaction with the
helicase)1 but also with
itself and the PNPase. However, under the same conditions, no
interactions between the RhlB and enolase or enolase and PNPase (Fig.
1C) were detected. We quantified the Individually Specific Interactions between RhlB RNA Helicase,
PNPase, and RE12 Are through the C-terminal Region of RhlB--
To
identify the regions of RhlB RNA helicase directly responsible for its
protein-protein interactions, several truncated RhlB derivatives were
constructed as described under "Experimental Procedures," based on
the predicted conserved sequence and functional motifs of a DEAD box
helicase family (36, 37). As shown in Fig.
2A, RhlB1 consists of the
ATPase motif. RhlB2 consists of the RhlB RNA helicase region including
helicase and RNA interaction motifs. RhlB3 consists of the C-terminal
region and lacks any conserved motifs. RhlB12, RhlB13, and RhlB23
consist of RhlB1 and -2, RhlB1 and -3, and RhlB2 and -3, respectively.
In the E. coli two-hybrid system, both color phenotypes on
MacConkey/maltose plates and
The Real-time Detection of Protein-Protein Interaction by BIAcore
Analyses Reveals Specific, Direct Binding of RhlB RNA Helicase to
Itself and PNPase but Not to Enolase--
To examine direct
interactions between components of the degradosome, we used purified
component proteins and an in vitro real-time binding assay
using BIAcore apparatus (38). The unbound and FLAG-tagged protein
components PNP, RhlB RNA helicase, and enolase were purified from BZ99
(an E. coli mutant containing only the RNase E N-terminal
region (amino acid residues 1-602)), and their enzymatic activities
were confirmed, as described under "Experimental Procedures." Fig.
3A shows Coomassie Blue
staining of high purity purified proteins. For the BIAcore surface
plasmon resonance analyses (38), RhlB RNA helicase was first
immobilized on a CM5 sensor chip, and different concentrations of three
distinct native degradosome proteins, enolase, RhlB helicase, and
PNPase (Fig. 3, B-D), were individually injected over the
chip surface, and the sensorgrams recorded. In these analyses, the
sensorgrams, in general, show an initial rapid increase in response
units upon protein injection followed by the association of injected
proteins with molecules coated on the chip and a slower association
phase characteristic of reversible protein interaction, resulting in a
plateau indicative of interacting saturation or equilibrium (Fig. 3).
After a change of buffer, there is a rapid decrease in response
unit/signal followed by the dissociation phase. As shown in Fig.
3B, the sensorgrams did not detect any RhlB-enolase interaction, whereas significant interaction responses were detected for RhlB with itself and with PNPase (Fig. 3, C and
D, respectively). These results indicated that RhlB helicase
was able to directly interact with PNPase and itself but not with
enolase (Fig. 3B). The sensorgrams (Fig. 3, C-E)
also indicated that the affinity of the RhlB RNA helicase interaction
with itself was higher than that of its interaction with PNPase (only
12.5 nM is shown in Fig. 3E). These results are
consistent with the results of our E. coli two-hybrid assay
(Fig. 1B).
We further addressed whether the non-monomeric form of RhlB RNA
helicase can directly interact with PNPase. As shown by sensorgrams, purified RhlB RNA helicase interacted with itself, after which these
self-associated molecules of RhlB RNA helicase were still able to bind
to PNPase (Fig. 4). Thus, these results
illustrated that under the experimental conditions carried out, a
dimeric (or multimeric) form of RhlB RNA helicase can physically bind to PNPase.
RhlB RNA Helicase Shows Functional Interaction with PNPase
Independent of RNase E--
In contrast to previously reported
degradosome studies, which used a mini-degradosome complex containing
the C-terminal region of RNase E, here we assessed the possibility of
functional interaction between RhlB and PNPase in the absence of RNase
E. The unwinding activity of RhlB RNA helicase was examined using
classical gel electrophoresis assays (39), as described under
"Experimental Procedures." Both single-stranded RNA
oligonucleotides were labeled with 32P and annealed to form
a double-stranded RNA substrate (labeled as double-stranded RNA). As
shown in Fig. 5 RhlB RNA helicase was
able to unwind duplex RNA (Fig. 5, lanes 2-7), and this
effect was RhlB-concentration-dependent (compare
lanes 6 and 7 versus 5). As
a result of the unwinding activity of RhlB, a decrease in
double-stranded RNA and an increase in single-stranded RNA were
observed. In the absence of RhlB under the same reaction conditions
PNPase quickly degraded single stranded RNA (labeled as long or short)
but not double-stranded RNA (Fig. 5, lanes 8-10 versus 11). Collectively, these experiments
indicate that the unwinding ability of RhlB enables PNPase to rapidly
degrade duplex double-stranded RNA.
Here we report that RNA degradosome protein component RhlB
interacts with PNPase independently of RNase E, which up to this point
has been believed to be the necessary building scaffold for
interactions among degradosome proteins. RhlB and PNPase interact through the C-terminal region of RhlB RNA helicase enabling PNPase to
degrade double-stranded RNA substrates. We also show that monomers of
RhlB interact among themselves, although at a site distinct from the
one that interacts with PNPase. It is interesting that Rh1B binds more
strongly to itself and to an RNase E fragment than it does to PNPase.
There are a number of reasons, both biochemical and cellular, that may
explain this result. First, the interaction surface (between the two
proteins) may be more favorable for RhlB binding to RNase E than for
the RhlB-PNPase interaction. Second, from a cellular perspective, RNase
E is a membrane-associated protein of relatively low abundance compared
with the highly abundant cytoplasmic PNPase; PNPase is
present in E. coli in excess of RNase E and has
been detected in cells unattached to the RNase E scaffold (27). If the
RhlB-PNPase interaction is stronger than the RhlB-RNase E interaction,
this may affect the availability of RhlB to bind to RNase E and
form degradosome complexes. This in turn could compromise RNA
degradation via a membrane-associated degradosome response.
Our results indicate that to degrade double-stranded RNA, PNPase would
need to recruit Rh1B helicase to open the double-stranded RNA secondary
structure, thereby allowing the exonuclease action of PNPase to degrade
the RNA, lending support to the hypothesis that DEAD box RhlB helicase
function is necessary for degradosome-mediated RNA decay. Our findings
also suggest that multimers of Rh1B RNA helicase may form and bind
directly to molecules of PNPase, independently of RNase E, to form
mini-degradation engines in E. coli capable of removing RNA
degradation intermediates (products) that have secondary structures.
A 3'-5' exonuclease complex, termed the "exosome," has been
identified in yeast cells (41, 42) with exosome protein component equivalents also discovered in mammalian cells (43). Recent modeling
experiments (44) suggest that exosomes may use the 3'-5' exonuclease
PNPase at their core. Although, E. coli degradosome models
have considered RNase E to be at the core of this bacterial ribonucleolytic complex (25), our discovery of PNPase-based complexes
that do not include RNase E, provides a molecular basis for alternative
pathways of E. coli RNA decay independent of RNase E-mediated endonucleolytic cleavages. Recent evidence suggesting that
functional inactivation of chloramphenicol acetyltransferase mRNA
in E. coli cell extracts occurs by a mechanism other than endonucleolytic cleavage (45), and earlier findings showing that
mutations of the RNase E-encoding rne gene affects chemical half-life but not functional decay (13) are consistent with this view.
It has been suggested that enolase is the Far Western and yeast two-hybrid analyses of interactions between RNase
E truncated polypeptides and RhlB have shown that RhlB binds directly
to both the central (500-752) and C-terminal (734-1061) regions of
RNase E, suggesting that important determinants for the binding are
located in the small overlap region (734-752) of RNase E segments;
other interactions among degradosome protein components were not
detected (25). Using E. coli two hybrid and BIAcore
biosensor analyses, we detected RhlB self-interaction and direct
binding to PNPase, in addition to its interaction with the RNase E
(684-784). These results indicate that the protein conformation of
various fusion products of RhlB and PNPase used for yeast and E. coli two-hybrid analyses differ and thus that it is appropriate to
explore multiple approaches for the study of protein-protein
interactions. Use of the BIAcore biosensor to analyze real time
interactions among degradosome protein components and between protein
component and RNA substrates to determine their kinetic constants may
help to elucidate the mechanism by which the dynamic processes of
degradosome assembly and RNA degradation interrelate.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
trpE5 mukB106 smbB199
(
))(28) and DHP1 (F
glnV44(AS) recA1 endA1
gyrA96(Nalr) thi1 hsdR17 spoT1 rfbD1 cya)(29) were used to
overexpress degradosome protein components and study in vivo
protein-protein interactions, respectively. BZ99 contains a truncated
rne gene that encodes amino acids 1-602 of the Rne protein.
DHP1 is an adenylate cyclase (cya)-deficient derivative of
DH1 (30). The plasmid pGP1-2, containing the T7 RNA polymerase gene
under the control of a temperature-sensitive bacteriophage repressor (31), was used to conditionally induce the synthesis of
individual proteins. A PCR-generated EcoRI-XhoI
fragment encoding full-length PNPase or RhlB RNA helicase and a
NdeI-BamHI fragment encoding full-length enolase,
were individually inserted into FLAG-containing vectors (the
corresponding plasmids were termed pflag-PNP, pflag-RhlB and
pflag-ENO). Proteins were purified using an M2 affinity column as
described (3). To study protein-protein interactions, a series of pT18-
or pT25-based vectors (29) encoding genes of interest were constructed:
pT25RE12, pT25PNP, pT25ENO, pT25RhlB, pT25RhlB1, pT25RhlB2, pT25RhlB3,
pT25RhlB12, pT25RhlB13, and pT25RhlB23. These constructs were generated
by the insertions of a PCR-generated PstI-BamHI
fragment encoding truncated peptides of Rne (i.e. RE12:
residues 684-784), full-length PNPase, enolase, RhlB RNA helicase and
some distinct motifs of RhlB polypeptide (i.e. RhlB1:
residues 1-194; RhlB2: residues 194-368; RhlB3: residues 351-421;
RhlB12: residues 1-368; RhlB13: residues 1-194, and 368-421; RhlB23:
residues 194-421, respectively), into the pT25-based vector. Plasmids
pPNPT18, pRhlBT18, and pENOT18, were generated by the insertions of a
PCR-generated XhoI-HindIII fragment encoding
full-length PNPase, RhlB, helicase and enolase, respectively, into the
pT18-based vector (29). All plasmid constructs were partially sequenced to confirm correct open reading frames.
-galactosidase activity on bacterial suspensions as previously described (32). For individual protein assays, log-phase 30 °C bacterial cultures at cell densities of ~2-5 × 108 cells/ml
(A600 of ~0.3-0.7) were used. Under
identical experimental condition, we also carried out -galactosidase
activity measurements, resulting from the interaction between T25-zip
and T18-zip leucine zipper motifs (29) to normalize individual protein
assays. For individual pairs of protein-protein interactions,
triplicate samples or more were studied.
-32P]ATP as described previously (35). The labeled
RNA strands were hybridized to form duplex RNA substrates made by
combining "short" and "long" RNA in a 1:1.5 ratio. The
excess of long RNA ensured that the majority of the labeled RNA formed
duplex RNA substrates. RNA annealing was performed by adding equal
volumes of 2× hybridization buffer (40 mM HEPES-KOH, pH
7.6, 1 M NaCl, 0.2% SDS, 2 mM EDTA) to long
and short RNA mixtures, in which samples were heated to 95 °C for 5 min and then slowly cooled to room temperature. The duplex RNAs (25 fmol) were incubated with purified RhlB RNA helicase alone or with
mixtures of PNPase and RhlB RNA helicase at 37 °C in final reaction
volumes of 20 µl containing 20 mM HEPES-KOH, pH 7.5, 70 mM KCl, 2 mM dithiothreitol, 0.1 mg of bovine
serum albumin, 1 mM magnesium acetate, and 3 mM
ATP. Each reaction was terminated by adding a 2 µl of stop solution
(2.2% Ficoll 400, 0.11 M EDTA, 1.1% SDS, 0.275%
bromphenol blue, 0.275% xylene cyanol FF). Samples were separated on a
16% polyacrylamide native gel (19:1 bisacrylamide) in 1× TBE. Labeled RNAs were visualized by autoradiography and quantified by LAS-1000 plus
(Fuji Film).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity in individual transformants grown to log-phase culture to
determine the efficiency of adenylate cyclase functional
complementation, representative of the strength of functional
interactions between two protein components (29). As shown in Fig.
1B, the strength of functional interactions is similar
between RhlB RNA helicase-RE12 and RhlB-RhlB RNA helicases and both are
stronger (about 4-fold) than that of the RhlB RNA helicase-PNPase
interaction. Consistently, the -galactosidase activity assay
indicated a lack of any interaction between enolase and the two
degradosome component proteins, RhlB and PNPase (Fig. 1, B
and C).
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Fig. 1.
Degradosome protein-protein
interactions as demonstrated by the E. coli two-hybrid
system. The various chimeric degradosome protein-constructs used
for individual complementation assays are shown. MacConkey/maltose agar
plate assays and -galactosidase activity assays were performed as
described under "Experimental Procedures." A,
C, chimeric degradosome protein-construct pairs
T25RhlB-RE12T18, T25RhlB-RhlBT18, and T25RhlB-PNPT18, but not isolated
individual chimeric proteins, revealed a positive interaction as shown
by the red colony phenotype. The positive control
T25Zip-ZipT18's interaction is also shown. No interactions between
construct pairs T25RhlB-EnoT18 and T25Eno-PNPT18 were detected. A
reversed chimeric construct of the Eno-PNP construct pair
(i.e. T25PNP-EnoT18) confirmed a lack of protein-protein
interaction between enolase and PNPase. B, -galactosidase
assay used to study the relative strength of protein-protein
interactions. The chimeric Zip-dimer was used as positive control,
(reaction activity was normalized to 100% and compared with other
chimeric pair interactions) as shown. The results represent the mean
value from at least three independent cultures: RE12, fragment of RNase
E, which is sufficient for an interaction with RhlB RNA helicase; PNP,
PNPase; RhlB, RhlB RNA helicase; and ENO, enolase. All plasmid
constructs encoding individually chimeric proteins are described under
"Experimental Procedures."
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Fig. 2.
Specific interaction between RhlB RNA
helicase and PNPase is mediated via the C-terminal region of RhlB, a
region not adequate by itself for RhlB-RhlB self-interaction.
A, a schematic diagram shows the conserved regions of the
DEAD box helicase family as regards E. coli RhlB RNA
helicase and its truncated derivatives RhlB1, RhlB2, RhlB3, RhlB12,
RhlB13, and RhlB23. Amino acid residues of individual derivatives are
indicated. B-J, the E. coli two-hybrid assay was
performed to study chimeric protein interactions as described in Fig. 1
in which bacteria were grown on either MacConkey/maltose plates
(B, E, H) or were cultured in the
presence of isopropyl -D-thiogalactoside plus
appropriate antibiotics (D, G, J)
(Karimova et al., Ref. 29). The chimeric protein interaction
pairs are also shown (C, F, I).
-galactosidase assays were used to study the strength of
protein-protein interactions. The positive control (T25Zip-ZipT18) and
the method used for comparison of protein-protein interaction strength
are described in the legend to Fig. 1. The results showed there is no
self-interaction detected between various RhlB-truncated derivatives
except for a full-length RhlB-RhlB interaction; the interaction between
RhlB and PNPase is associated with the C-terminal region of RhlB.
-galactosidase activity assays showed that RhlB23 was sufficient to interact with RE12 and PNPase (Fig. 2,
B-D, E-G, respectively). Interestingly, the
region of RhlB sufficient for interaction with PNPase and RE12 fails to
interact with itself. RhlB RNA helicase self-interaction requires
full-length RhlB RNA helicase (Fig. 2, H and
J).
-galactosidase activity assay indicates that the strength of
interaction between RhlB23 and RE12 is a little weaker than that
between full-length RhlB RNA helicase and RE12 (Fig. 2D). In
contrast, the strength of interaction between RhlB23 and PNPase is
similar to that between full-length RhlB RNA helicase and PNPase (Fig.
2G).
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Fig. 3.
Kinetic analyses of degradosome
protein component interactions. A, Coomassie Blue
staining of purified proteins PNPase, RhlB RNA helicase, and enolase
separated by 8% SDS-PAGE. Protein purification is described under
"Experimental Procedures." B-D, kinetic data set
collected for individual analytes (i.e. degradosome protein
components) binding to an RhlB-surface chip. Degradosome component
proteins at various concentrations, as indicated, were injected over
the RhlB surface. Injections were carried out as described under
"Experimental Procedures." Normalized binding responses for enolase
(B), RhlB RNA helicase (C), and PNPase
(D) were reproducible for individual injections (data not
shown). Individual injections were performed at least four to five
times. E, comparison of the kinetic data for RhlB RNA
helicase, enolase, and PNPase, respectively, injected over the RhlB
surface chip. No response was detected for enolase-RhlB, however, a
"strong" response was detected for PNPase-RhlB, and an even
stronger response for RhlB-RhlB interaction reaction. A detailed
account for the basic shape of the graph can be found under
"Experimental Procedures" and "Results."
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Fig. 4.
Self-interacted form of RhlB-RhlB RNA
helicase binds to PNPase. The distinct protein analytes and their
concentrations are indicated on the sensorgram. RhlB RNA helicase was
injected over the RhlB chip surface; after the dissociation phase,
injection of the second analyte (i.e. the PNPase) over the
RhlB-RhlB surface was carried out to generate the data presented in
this sensorgram.
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Fig. 5.
RhlB unwinding activity is necessary for
PNPase to degrade double-stranded RNAs. Both synthesized long (22 mer) and short (11 mer) RNA substrates were labeled at their 5'-ends
with -P32 as indicated with asterisks.
Vertical lines between two RNA substrates indicate the
double-stranded RNA region. The RNA sequences are described under
"Experimental Procedures." Lane 1 contains a heat
denature control showing separated single strand short and long RNA.
Lanes 2-7 are the time courses showing RhlB RNA unwinding
activities. Lanes 8-10 are the time courses showing PNPase
exoribonuclease activity to degrade single-stranded RNA but not
double-stranded RNA. Lane 11 is the time course of the
simultaneous reaction between RhlB and PNPase that degrades
double-stranded RNA substrates. The nucleotide sequences of both short
and long RNA substrates are described under "Experimental
Procedures." +, presence of enzyme (0.63 µM); 
,
absence of enzyme; 2× and 3×, the concentration of enzyme used (1.26 and 1.89 µM, respectively). RhlB, FLAG-RhlB RNA helicase;
PNP, FLAG-PNPase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of
32 form PNPase (PNPase is the subunit) (4), although no experimental proof has been forthcoming.
However, our results indicate that RhlB RNA helicase, not enolase, is a
native component of the PNPase complex. We detected no binding between
enolase and PNPase (a similar conclusion also has been discussed by
Kühnel and Luisi (Ref. 46; see "Discussion")). The yeast
two-hybrid system also failed to detect any interaction between the two
proteins (25). Furthermore, we have shown in this study that RhlB
contains as ATP-dependent RNA unwinding ability (not
demonstrated prior to this study) and that this ATP-activated unwinding
activity enables PNPase to degrade double-stranded RNA; whether the
binding of RhlB to PNPase regulates PNPase catalytic activity or vise
versa remains to be clarified.
| |
ACKNOWLEDGEMENTS |
|---|
Plasmid-based vectors pT18 and pT25 and E. coli strains were kindly provided by Dr. D. Ladant, Institut Pasteur, Paris Cedex, France. We thank our English Editing consultant Dr. K. Deen for his constructive comments concerning this manuscript and Drs. S. N. Cohen and C. Wang for their critical comments on this manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by an intramural fund from the Academia Sinica and by the Research Grant NSC90-2321-B-001-005 of Frontier Sciences from the National Science Council, Taiwan, R. O. C. (to S. L.-C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of postdoctoral fellowship from the National Science Council, Taiwan, R.O.C.
** To whom correspondence should be addressed. Tel.: 886-2-2789-9218; Fax: 886-2-27826085; E-mail: mbsue@ccvax.sinica.edu.tw.
Published, JBC Papers in Press, August 13, 2002, DOI 10.1074/jbc.M206618200
1 G.-G. Liou, H.-Y. Chang, C.-S. Lin, and S. Lin-Chao, unpublished data.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Carpousis, A. J., Van Houwe, G., Ehretsmann, C., and Krisch, H. M. (1994) Cell 76, 889-900[CrossRef][Medline] [Order article via Infotrieve] |
| 2. | Py, B., Causton, H., Mudd, E. A., and Higgins, C. F. (1994) Mol. Microbiol. 14, 717-729[Medline] [Order article via Infotrieve] |
| 3. |
Miczak, A.,
Kaberdin, V. R.,
Wei, C. L.,
and Lin-Chao, S.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
3865-3869 |
| 4. | Py, B., Higgins, C. F., Krisch, H. M., and Carpousis, A. J. (1996) Nature 381, 169-172[CrossRef][Medline] [Order article via Infotrieve] |
| 5. | Carpousis, A. J., Vanzo, N. F., and Raynal, L. C. (1999) Trends Genet 15, 24-28[CrossRef][Medline] [Order article via Infotrieve] |
| 6. | Coburn, G. A., and Mackie, G. A. (1999) Prog. Nucleic Acids Res. Mol. Biol. 61, 55-108 |
| 7. | Grunberg-Manago, M. (1999) Annu. Rev. Genet. 33, 193-227[CrossRef][Medline] [Order article via Infotrieve] |
| 8. | Nicholson, A. W. (1999) FEMS Microbiol. Rev. 23, 371-390[CrossRef][Medline] [Order article via Infotrieve] |
| 9. | Rauhut, R., and Klug, G. (1999) FEMS Microbiol. Rev. 23, 353-370[CrossRef][Medline] [Order article via Infotrieve] |
| 10. | Régnier, P., and Arraiano, C. M. (2000) BioEssays 22, 235-244[CrossRef][Medline] [Order article via Infotrieve] |
| 11. | Steege, D. A. (2000) RNA 6, 1079-1090[Abstract] |
| 12. |
Apirion, D.
(1978)
Genetics
90,
659-671 |
| 13. | Ono, M., and Kuwano, M. (1979) J. Mol. Biol. 129, 343-357[CrossRef][Medline] [Order article via Infotrieve] |
| 14. | Jain, C., Deana, A., and Belasco, J. G. (2002) Mol. Microbiol. 43, 1053-1064[CrossRef][Medline] [Order article via Infotrieve] |
| 15. | Ghora, B. K., and Apirion, D. (1978) Cell 15, 1055-1066[CrossRef][Medline] [Order article via Infotrieve] |
| 16. |
Lin-Chao, S.,
Wei, C.-L.,
and Lin, Y.-T.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12406-12411 |
| 17. | Mudd, E. A., Krisch, H. M., and Higgins, C. F. (1990) Mol. Microbiol. 4, 2127-2135[Medline] [Order article via Infotrieve] |
| 18. | Lin-Chao, S., and Cohen, S. N. (1991) Cell 65, 1233-1242[CrossRef][Medline] [Order article via Infotrieve] |
| 19. |
Régnier, P.,
Grunberg-Manago, M.,
and Portier, C.
(1987)
J. Biol. Chem.
262,
63-68 |
| 20. | Kalman, M., Murphy, H., and Cashel, M. (1991) New Biol. 3, 886-895[Medline] [Order article via Infotrieve] |
| 21. |
Spring, T. G.,
and Wold, F.
(1971)
J. Biol. Chem.
246,
6797-6802 |
| 22. | Blum, E., Py, B., Carpousis, A. J., and Higgins, C. F. (1997) Mol. Microbiol. 26, 387-398[CrossRef][Medline] [Order article via Infotrieve] |
| 23. |
Bessarab, D. A.,
Kaberdin, V. R.,
Wei, C.-L.,
Liou, G.-G.,
and Lin-Chao, S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3157-3161 |
| 24. |
Kaberdin, V. R.,
Miczak, A.,
Jakobsen, J. S.,
Lin-Chao, S.,
McDowall, K. J.,
and von Gabain, A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11637-11642 |
| 25. |
Vanzo, N. F., Li, Y. S., Py, B.,
Blum, E.,
Higgins, C. F.,
Raynal, L. C.,
Krish, H. M.,
and Carpousis, A. J.
(1998)
Genes Dev.
12,
2770-2781 |
| 26. |
Coburn, G. A.,
Miao, X.,
Briant, D. J.,
and Mackie, G. A.
(1999)
Genes Dev.
13,
2594-2603 |
| 27. |
Liou, G.-G.,
Jane, W.-N.,
Cohen, S. N.,
Lin, N.-S.,
and Lin-Chao, S.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
63-68 |
| 28. |
Kido, M.,
Yamanaka, K.,
Mitani, T.,
Niki, H.,
Ogura, T.,
and Hiraga, S.
(1996)
J. Bacteriol.
178,
3917-3925 |
| 29. |
Karimova, G.,
Pidoux, J.,
Ullmann, A.,
and Ladant, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5752-5756 |
| 30. | Hanahan, D. (1983) J. Mol. Biol. 166, 557-580[Medline] [Order article via Infotrieve] |
| 31. |
Tabor, S.,
and Richardson, C. C.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
1074-1078 |
| 32. | Miller, J. H. (1972) Experiments in Molecular Genetics , pp. 352-355, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
| 33. |
Lin-Chao, S.,
Wong, T. T.,
McDowall, K. J.,
and Cohen, S. N.
(1994)
J. Biol. Chem.
269,
10797-10803 |
| 34. |
McDowall, K. J.,
Lin-Chao, S.,
and Cohen, S. N.
(1994)
J. Biol. Chem.
269,
10790-10796 |
| 35. | McDowall, K. J., Kaberdin, V. R., Wu, S. W., Cohen, S. N., and Lin-Chao, S. (1995) Nature 374, 287-290[CrossRef][Medline] [Order article via Infotrieve] |
| 36. | de la Cruz, J., Kressler, D., and Linder, P. (1999) Trends Biochem. Sci. 24, 192-198[CrossRef][Medline] [Order article via Infotrieve] |
| 37. | Lüking, A., Stahl, U., and Schmidt, U. (1998) Crit. Rev. Biochem. Mol. Biol. 33, 259-296[CrossRef][Medline] [Order article via Infotrieve] |
| 38. | Hutchinson, A. M. (1995) Mol. Biotechnol. 3, 47-54[Medline] [Order article via Infotrieve] |
| 39. | Matson, S. W., and Bean, D. W. (1995) Methods Enzymol. 262, 389-405[Medline] [Order article via Infotrieve] |
| 40. | Deleted in proof |
| 41. | Mitchell, P., Petfalski, E., Shevcheno, A., Mann, M., and Tollervey, D. (1997) Cell 91, 457-466[CrossRef][Medline] [Order article via Infotrieve] |
| 42. | Mitchell, P., and Tollervey, D. (2000) Nat. Struct. Biol. 7, 843-846[CrossRef][Medline] [Order article via Infotrieve] |
| 43. | Chen, C. Y., Gherzi, R., Ong, S. E., Chan, E. L., Raijmakers, R., Pruijn, G. J., Stoecklin, G., Moroni, C., Mann, M., and Karin, M. (2001) Cell 107, 451-464[CrossRef][Medline] [Order article via Infotrieve] |
| 44. | Aloy, P., Ciccarelli, F. D., Leutwein, C., Gavin, A. C., Superti-Furga, G., Bork, P., Böttcher, B., and Russell, R. B. (2002) EMBO Rep. 3, 628-635[CrossRef][Medline] [Order article via Infotrieve] |
| 45. |
Lee, K.,
and Cohen, S. N.
(2001)
J. Biol. Chem.
276,
23268-23274 |
| 46. | Kühnel, K., and Luisi, B. F. (2001) J. Mol. Biol. 313, 583-592[CrossRef][Medline] [Order article via Infotrieve] |
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