From the Department of Biochemistry, Case Western Reserve
University School of Medicine, Cleveland, Ohio 44106-4935 and
Isis Pharmaceuticals, Inc.,
Carlsbad, California 92008
Received for publication, August 18, 2000, and in revised form, December 28, 2000
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ABSTRACT |
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Eukaryotic initiation factor (eIF) 4A is the
archetypal member of the DEAD box family of RNA helicases and is
proposed to unwind structures in the 5'-untranslated region of mRNA
to facilitate binding of the 40 S ribosomal subunit. The helicase
activity of eIF4A has been further characterized with respect to
substrate specificity and directionality. Results confirm that the
initial rate and amplitude of duplex unwinding by eIF4A is dependent on the overall stability, rather than the length or sequence, of the
duplex substrate. eIF4A helicase activity is minimally dependent on the
length of the single-stranded region adjacent to the double-stranded region of the substrate. Interestingly, eIF4A is able to unwind blunt-ended duplexes. eIF4A helicase activity is also affected by
substitution of 2'-OH (RNA) groups with 2'-H (DNA) or 2'-methoxyethyl groups. These observations, taken together with results from
competitive inhibition experiments, suggest that eIF4A may interact
directly with double-stranded RNA, and recognition of helicase
substrates occurs via chemical and/or structural features of the
duplex. These results allow for refinement of a previously proposed
model for the mechanism of action of eIF4A helicase activity.
The initiation of protein synthesis in eukaryotic systems is an
intricate process involving at least 12 protein factors that work in
concert to bring the mRNA, the initiating methionyl-tRNA, and the
40 S and 60 S ribosomal subunits together to form an 80 S complex
capable of peptide chain elongation (see Refs. 1-3 for reviews on
translation initiation). In this process, eukaryotic initiation factor
(eIF)1 4A works in
conjunction with eIF4B, eIF4F, and eIF4H to promote the binding of
mRNA to the 40 S ribosomal subunit (1, 3, 4). eIF4A is proposed to
function in this important regulatory step by unwinding secondary
structure in the 5'-untranslated region of the mRNA, thus
facilitating the binding of the 40 S ribosomal subunit to the mRNA
and allowing for subsequent scanning to the initiator AUG codon.
eIF4A is the archetype of the DEAD box family of proteins (5).
DEAD box (and related DEXH box) proteins contain
eight highly conserved amino acid sequence motifs and have been
implicated in virtually every cellular process involving RNA unwinding
and/or rearrangement. These include transcription, ribosomal
biogenesis, pre-mRNA splicing, RNA export, translation, and RNA
degradation (6, 7). In vitro analysis of this family of
proteins has demonstrated that all members possess
RNA-dependent ATPase activity, and many are capable of
melting short RNA duplex structures in an ATP-dependent
manner (8-13). In addition to providing the energy for unwinding RNA
duplexes, it has been postulated that the ATPase activities of
DEAD/DEXH proteins may function in part as kinetic proofreading devices and maintain fidelity in complex biological processes (14, 15). Furthermore, many viruses also encode DEAD/DEXH proteins that exhibit RNA- or
DNA-dependent ATPase activities and
ATP-dependent RNA or DNA unwinding activities, which are
presumably needed for viral replication (16-22). Recently, Jankowsky
et al. (23) have elegantly demonstrated that the NPH-II
protein of vaccinia virus is able to function as a processive and
directional RNA helicase.
The eight conserved motifs of eIF4A span 400 amino acid residues and
represent the core sequence of DEAD/DEXH proteins in the
superfamily II group of helicases (5, 6, 24). Extensive mutational
analyses of the conserved regions of eIF4A and other DEAD/DEXH proteins have demonstrated that they are important
for ATP binding, ATP hydrolysis, RNA binding, RNA unwinding, and
coupling of these different activities (17, 25-30). Two groups have
presented partial crystal structures of yeast eIF4A (31, 32). Both show that the three-dimensional fold, strand topology, and the orientation of conserved motifs in the N-terminal portion of eIF4A are strikingly similar to domain I of the HCV NS3, PcrA, and Rep helicases (31, 32).
Recently, the crystal structure of the C-terminal portion eIF4A has
been solved and again shows that tertiary structure and positions of
the conserved motifs in eIF4A have the same topology as the equivalent
domains of the Rep, PcrA, and HCV NS3 helicases (33). Such similarities
strongly suggest a common structural and mechanistic theme among these
helicases (34).
eIF4A has been the subject of intense biochemical study. A detailed
kinetic and thermodynamic analysis of the RNA-activated ATPase activity
of eIF4A has been performed, which showed that the binding of RNA and
ATP to eIF4A is coupled (35). These data, along with limited
proteolysis experiments, suggest that eIF4A undergoes a series of
ligand-dependent conformational changes as it binds its
substrates (RNA and ATP), hydrolyzes ATP, and releases products (36).
The effect of changing oligonucleotide length, backbone composition,
and identity of the 2'-substituent of the ribose moiety has been
investigated and shown to alter the nucleic acid binding and ATPase
activities of eIF4A (37).
Previously, we have demonstrated that eIF4A functions alone as an
ATP-dependent RNA helicase (8). Results indicated that the
initial rate of eIF4A-dependent unwinding decreased as the stability of the duplex is increased and that eIF4A helicase activity is nonprocessive (8). A simple model of unwinding was proposed suggesting that eIF4A affects strand separation by unwinding a small
number of base pairs, consequently making the duplex behave as a
shorter, less stable duplex in a thermodynamic sense. If there is
sufficient destabilization of the duplex by eIF4A, complete stand
separation occurs (less stable duplex substrates). Conversely, if the
duplex is not sufficiently destabilized by eIF4A, the partially unwound
strands reanneal (i.e. snap back), and complete strand separation does not occur (more stable duplex substrates) (8). In
addition, the helicase activity of eIF4A may be stimulated by either
eIF4B or eIF4H or if eIF4A exists as part of the eIF4F complex (4, 8,
38). However, it has not been determined if there is a minimal size of
the single-stranded region adjacent to the duplex region of the
substrate needed to promote helicase activity or if eIF4A alone may
function bidirectionally. Furthermore, the effect of changing the
2'-substituents of the ribose sugars or the phosphate backbone of the
duplex on the helicase activity of eIF4A is unknown. In a continuing
effort to understand the mechanism by which eIF4A and other
DEAD/DEXH proteins unwind nucleic acid duplexes, an
investigation of the helicase activity of eIF4A with respect to duplex
substrate specificity was performed.
Materials
Reagents were purchased from the following suppliers: rabbit
reticulocyte lysate from Green Hectares, Oregon, WI; ATP and BSA from
Sigma; [ Methods
Purification of eIF4A from Rabbit Reticulocyte
Lysate--
Purification of eIF4A follows the standard procedure used
to purify protein translation initiation factors that have been previously published by this laboratory (39, 40).
DNA and RNA Oligonucleotides--
DNA oligonucleotides
synthesized by the Molecular Biology Core Laboratory, Case Western
Reserve University, were oligonucleotide purification
cartridge-purified and stored in distilled H2O. DNA oligonucleotides synthesized by IDT, Inc., were polyacrylamide gel
electrophoresis-purified, lyophilized, and resuspended in distilled H2O. RNA oligonucleotides synthesized by
Cybersyn were polyacrylamide gel electrophoresis-purified, lyophilized,
and resuspended in distilled H2O. RNA
oligonucleotides synthesized by Dharmacon were deprotected per the
manufacturer's instructions, lyophilized, and resuspended in
distilled H2O. Quantitation of each oligonucleotide
was performed by UV spectroscopy, and a value of 33 µg per 1 A260 was used in determining concentration.
Integrity and proper size of each oligonucleotide was assessed by
32P-end-labeling each oligonucleotide (described below) and
analysis on a denaturing (7 M urea) 20% polyacrylamide gel
with known size standards.
Transcription of RNA--
R-44, -41SD, -41S-, -41S+, and -44L+
(see Table I) were synthesized by in vitro transcription
using T7 RNA polymerase. The templates for transcription were composed
of PTT-23 annealed to PTB-44, -41SD, -41S-, -41S+, or -44L+ (see Table
I). Transcription reactions were performed using Ambion's
MegashortscriptTM transcription kit per the manufacturer's
instructions and included 1 µl of [ 32P-End-labeling of Oligonucleotides--
Forty
picomoles of either R-10, -11, -12, -13, -14, -15, -13SD, -13S-, -13S+,
-16L+, and -13C or D-14, -15, -16, and -17 oligonucleotides (see Table
I) and 10 pmol of [ Helicase Substrates--
Duplexes used in the helicase reactions
are made by combining long oligonucleotides with appropriate
complementary 32P-labeled short oligonucleotides (see Table
I for individual oligonucleotide lengths and sequences and figures for
duplexes used in specific experiments) in a 1.25:1 ratio, respectively. This excess of long strand is to ensure that essentially all of the
labeled short oligonucleotide is in the duplex species. The complementary strands are combined in 1× hybridization buffer (1×
Tris-EDTA (TE) plus 100 mM KCl), and the concentration of duplex was 0.5 pmol/µl. Samples were heated to 95 °C for 5 min and
then slow-cooled to 4 °C over 90 min (0.1 °C/5 s) using a programmable thermocycling instrument. Under these conditions, ~90-95% of the labeled bottom (short) strand hybridizes to the top
(long) strand. The duplex was stored at dsRNA and dsDNA Competitor Duplexes--
The R-17-3'/R-17C and
D-20/D-20C duplexes used in the competition assay (Fig. 4) were
prepared as above except the R-17-3' and R-17C or D-20 and
D-20C oligonucleotides were combined in a 1:1 ratio at a
concentration of 105 or 89 pmol/µl, respectively. Control experiments
showed that no significant strand separation occurred at the lowest
final concentration of duplex used in the competition experiment (10 nM) over the time course of the reaction (data not shown).
Helicase Assay--
The helicase assay was performed as
described in detail previously (8). Unwinding of duplex substrates was
monitored by following displacement of the radiolabeled short strand
from the duplex. In general, 20-µl reactions contained 20 mM HEPES-KOH, pH 7.5 (final pH 7.2), 70 mM KCl,
2 mM dithiothreitol, 1 mg/ml BSA, and 1 mM
magnesium acetate (Buffer A). Buffer B' contained 20 mM
MES-KOH, pH 6.0, 15 mM KCl, 15 mM potassium
acetate, 2 mM dithiothreitol, 1 mg/ml BSA, and 1 mM magnesium acetate. The concentration of ATP was 1 mM; duplex concentration was 1.8-1.9 nM
(36-38 fmol), and eIF4A concentration was 0.8 µM (16 pmol), unless stated otherwise. All reactions were mixed in siliconized
tubes to minimize material sticking to tube walls and kept on ice
throughout the assembly process. Reactions were incubated at 35 °C
(unless otherwise stated) for the duration of the time course. In most
circumstances, eIF4A was the last component added to the reaction, just
before incubation at 35 °C; however, equivalent results were
obtained when duplex or competitor was added as the last component.
Reactions were terminated by adding 5 µl of a solution containing
50% glycerol, 2% SDS, 20 mM EDTA, and 0.05% bromphenol
blue and xylene cyanol dyes. The products of unwinding reactions were
analyzed by separation of displaced labeled short strand from duplex
species by electrophoresis on 15 or 18% native polyacrylamide gels
(19:1 acrylamide:bisacrylamide) at 4 °C for 2-3 h at 200 V in 1×
TBE (TBE, Tris borate/EDTA) buffer. Gels were pre-electrophoresed at
4 °C for 30 min. After electrophoresis, gels were scanned directly
using an Ambis radioanalytical scanner. Gels were then exposed to Kodak
X-Omat AR film at Quantitation of the Helicase Assay--
Quantitation of the
helicase assay was as described previously (8). In brief, quantitation
of counts/min in duplex and displaced/single-stranded RNA bands was
performed using the Ambis software. The degree of unwinding for each
reaction was determined by measuring the percent of counts/min in
duplex and displaced/single-stranded RNA bands using the following
formulas: % duplex = cpm duplex/(cpm duplex + cpm displaced
strand) and % displaced strand = cpm displaced strand/(cpm duplex + cpm displaced strand). This method of quantitation was used to
account for slightly different yields in total counts/min due to
variations in pipetting and gel loading steps. The total yield in
counts/min among different reactions did not vary by more than ±10%
of the average counts/min value of all the reactions in the given
experiment. Furthermore, all reagents and protein preparations used in
the assay were judged to be free of RNase and phosphatase
contamination. A single reaction without ( Data Treatment--
Treatment of data has been described
previously (8). All data reported are the average of 3-5 separate
experiments. Standard errors were less than ±10% in all experiments.
Plots and graphs were constructed using Prism software by GraphPad or
SlideWrite 4.0. Duplex stability (
Initial rates of unwinding duplex substrates by eIF4A were determined
by measuring the amount of unwinding in the linear (0-2 min) portion
of the reaction. Linear fits were applied, and the initial rate (in
fmol/min) was taken from the slope of this line (data not shown). The
amplitude (maximum % duplex unwound) of duplex unwinding was
determined by allowing the helicase reaction to proceed to completion
(time course experiments indicated that the reaction was complete in 15 min, data not shown and Ref. 8). This value is then used to make
comparisons with respect to the ability of eIF4A to unwind various
duplex substrates. This method of comparison is valid since a direct
linear correlation exists between the ln(amplitude of unwinding by
eIF4A) and duplex stability ( eIF4A Helicase Activity and Duplex Stability--
Previous studies
from this laboratory demonstrated the initial rate of duplex unwinding
by eIF4A decreased as the length and stability of the duplex
(
To examine the effect of duplex stability independent of duplex length
on the unwinding activity of eIF4A, three 13-bp duplexes of different
stabilities (
Previous studies demonstrated that the ln(initial rate of unwinding) by
eIF4A or the ln(initial rate of thermal
melting)2 shows a linear
correlation with duplex stability and suggests a model for how eIF4A
unwinds RNA duplexes (see Introduction and Ref. 8). Due to slight
differences in the length of the long strand of the duplex substrates
used in this study when compared with our previous work (R-44, 44 nt
versus R-1, 50 nt, respectively), the initial rates of
eIF4A-dependent unwinding and thermal melting were measured
for the R-44/R-10, -11, -12, -13, -14, and -15 duplexes (data not
shown). The ln(initial rate of unwinding or thermal melting) was
plotted versus duplex stability (Fig. 1B), and
the results are equivalent to those obtained previously (8).
To investigate a potential relationship between the maximum percentage
(amplitude) of eIF4A-dependent unwinding and duplex stability, the amplitudes of eIF4A-dependent unwinding and
thermal melting were measured for the R-44/R-10, -11, -12, -13, -14, and -15 duplexes (Table II and data not shown). The ln(amplitude of unwinding) and the ln(amplitude of thermal melting) for these substrates were plotted versus duplex stability (Fig.
1C, closed circles and open circles,
respectively). In addition, the ln(amplitude of unwinding) for the
duplexes substrates shown in Fig. 1A was also plotted
versus duplex stability (Fig. 1C, open squares). It can be seen from Fig. 1C that a linear correlation exists
between the ln(amplitude of unwinding or thermal melting) and duplex
stability. This relationship is very similar to that shown in Fig.
1B, and it is stressed that the values of the slopes are
equivalent in both Fig. 1, B and C (
Several conclusions may be drawn from the data presented in Fig. 1.
First, when taken together, the data presented in Fig. 1, A
and C, demonstrate that the overall stability of the duplex, rather than the length or nucleotide sequence, determines how efficiently eIF4A is able to unwind that duplex. Second, comparison of
Fig. 1, B with C, indicates that the relationship
between the amplitude of unwinding and duplex stability is similar to
that observed between the initial rate of unwinding and duplex
stability. Since the amplitude of unwinding may be linearly correlated
with duplex stability, measuring the amplitude provides an efficient means for comparing how effectively eIF4A is able to unwind duplexes with different stabilities and/or physical and chemical modifications. This is desirable since the amplitude of unwinding provides a more
accurate measure of eIF4A unwinding activity due to a higher signal to
noise ratio when compared with measurement of initial rates. It is
stressed that the amplitude measurements presented here are used only
to compare the overall macroscopic degree of unwinding various duplex
substrates by eIF4A and are not used to provide information concerning
the microscopic kinetic steps of the unwinding reaction.
Effect of the Length and Directionality of the
Single-stranded Region on eIF4A Helicase Activity--
Previously, it
was demonstrated (37) that the RNA-stimulated ATPase activity of eIF4A
was dependent on the length the ssRNA activator. These results were
consistent with former studies (49, 50) and together suggest a binding
site size of ~15 nucleotides (nt) for eIF4A. Therefore, it was of
interest to investigate if the helicase activity of eIF4A is also
affected by length of the single-stranded region adjacent to the
double-stranded region in a duplex substrate. In addition, previous
results demonstrated that a combination of either eIF4A + eIF4B or
eIF4F (eIF4A·eIF4E·eIF4G) + eIF4B was able to unwind RNA duplexes
in both 5'
To address how the helicase activity of eIF4A is affected by the length
and directionality of the single-stranded region adjacent to the duplex
region, a series of 13-bp duplex substrates with either 5'- or
3'-single-stranded regions ranging from 0 to 25 nt was designed (Fig.
2A). The sequences of both the
single-stranded and duplex regions are exactly the same for the 5' eIF4A Unwinding of Blunt-ended Duplexes and Interaction with
dsRNA--
Since the above result demonstrating that eIF4A could
unwind a blunt-ended RNA duplex was unexpected, additional experiments designed to explore this observation further were performed. To determine whether significantly different amounts of eIF4A were required for the unwinding of blunt-ended duplexes as compared with
duplexes containing a single-stranded region, the effect of eIF4A
concentration on unwinding activity was investigated. Fig.
3 shows the effect of increasing eIF4A
concentration using the R-38-5'/R-13 (25-nt single-stranded region) and
R-13/R-13C (blunt-ended) duplexes. The plot shows that the amplitude of
unwinding for both duplexes is linear with respect to the concentration of eIF4A (0-1.6 µM) and, further, that the amplitude of
unwinding the blunt-ended duplex is consistently 70% of the amplitude
of unwinding a duplex with a 5' single-stranded region of 25 nt. Furthermore, the initial rate of unwinding the blunt-ended duplex was
also ~70% of the initial rate of unwinding the duplex containing a
5' single-stranded region of 25 nt (0.8 µM eIF4A, data
not shown). Thus, the efficiency of unwinding a blunt-ended duplex by
eIF4A is only minimally decreased (
The results demonstrating that eIF4A can unwind a blunt-ended duplex
indicates that eIF4A may interact directly with dsRNA. To investigate
this possibility further, competitive inhibition experiments using
blunt-ended dsRNA were performed. If eIF4A interacts with a nonlabeled
dsRNA, then increasing concentrations of this species should abrogate
the unwinding of a labeled duplex substrate due to competitive
inhibition. The unwinding assay was performed using the R-41S-/R-13S-
duplex as a substrate, and a 17-bp blunt-ended dsRNA competitor
(R-17-3'/R-17C) was titrated into the reaction. It should be noted that
the dsRNA competitor's sequence is noncomplementary to the duplex
substrate, and control experiments showed the dsRNA competitor was too
stable to be unwound efficiently by eIF4A under the reaction conditions
used (data not shown). Since eIF4A binds to ssRNA, the competition
experiment was also performed with a 17-nt ssRNA (RNA-17C) to serve as
a control (data not shown and Fig. 3B). Fig. 3A
presents the experimental data for adding increasing concentrations of
the dsRNA competitor into the unwinding reaction. Fig. 3B
summarizes the results obtained from this series of experiments using
the ssRNA and dsRNA competitors. As expected, ssRNA served as a
competitive inhibitor to the helicase activity of eIF4A, with
half-maximal inhibition of helicase activity observed at 0.05 µM ssRNA. The blunt-ended dsRNA duplex also acted as an
inhibitor, although it was less effective when compared with the ssRNA
in competing for eIF4A, with half-maximal inhibition of helicase activity observed at 0.11 µM dsRNA (Fig. 3B).
This result implies that eIF4A binds directly to dsRNA. The ability of
ssDNA and dsDNA to inhibit the unwinding reaction was also
investigated. This will be addressed in more detail below.
Effect 2'-Ribose Group and Backbone Modifications on the Duplex
Unwinding Activity of eIF4A--
Although DEAD/DEXH
proteins are classified as RNA helicases, several members of this
family of proteins are also able to unwind RNA/DNA, DNA/RNA, and/or
DNA/DNA duplexes3 (16, 18,
20, 51, 52). Also, Peck and Herschlag (37) demonstrated the following.
(i) eIF4A is able to bind DNA with a similar affinity as RNA (although
DNA was unable to stimulate the ATPase activity of eIF4A). (ii)
Replacing the nonbridging oxygen atoms in phosphodiester (PO) linkages
with sulfur atoms to make phosphorothioate (PS) linkages had no
significant effect on the maximal stimulation of the ATPase activity of
eIF4A; however, there was a substantial increase in the affinity of
eIF4A for the modified oligonucleotide. (iii) Changing the 2'-OH
substituent of the ribose sugar to 2'-OCH3 reduced the
ATPase stimulation at least 100-fold without affecting the
oligonucleotide affinity. Thus, to investigate further the substrate
specificity and mechanism of the helicase activity of eIF4A,
experiments were performed in which the 2'-group of the ribose sugar
and/or the phosphate backbone of the duplex substrate was chemically modified.
First, the ability of eIF4A to unwind RNA/DNA, DNA/RNA, and DNA/DNA
duplex substrates was investigated. Several RNA/RNA duplexes were
compared with RNA/DNA (the single-stranded region is RNA), DNA/RNA (the
single-stranded region is DNA), and DNA/DNA duplexes of similar
sequence, length, and stability in the eIF4A-dependent unwinding assay. Presented in Fig. 5A is selected data of
eIF4A unwinding activity using RNA/RNA, RNA/DNA, DNA/RNA, and DNA/DNA duplex substrates. Table II lists the entire data set, including the
type of duplex, number of base pairs, calculated stability, and the
amplitude of unwinding by eIF4A. All substrates had a 5'-single-stranded region of at least 26 nt. As expected, eIF4A unwound
the RNA/RNA duplexes with the amplitude of unwinding correlating to the
stability of the duplex, and these results were equivalent to those
obtained previously (8). As evident from Fig. 5A and Table
II, eIF4A was also able to unwind RNA/DNA and DNA/RNA duplexes, and the
magnitude of unwinding was also dependent on the stability of the
duplex (i.e. as the stability of the duplex increased, the
amplitude of unwinding by eIF4A decreased). Furthermore, different types of duplexes with similar stabilities were unwound with similar efficiencies (e.g. compare R-44/R-13 with R-44/D-16 and
compare R-44/R-12 with D-44/R-17). In contrast, eIF4A was unable to
unwind either DNA/DNA duplex tested. These observations suggest eIF4A may interact with both RNA and DNA, since both RNA/DNA and DNA/RNA duplexes are unwound. However, since DNA/DNA duplexes are not unwound,
this indicates that eIF4A requires an RNA strand for helicase activity.
Next, we investigated the ability of eIF4A to unwind a duplex substrate
in which the long strand was RNA, but the short strand had either a
phosphorothioate (PS) backbone and/or a 2'-methoxyethyl (MOE)
modification. Fig. 5B presents a diagram of the backbone (PO
To investigate further potential interactions of eIF4A with ssDNA,
dsDNA, ssDNA-PS, or 2'-MOE, competition experiments similar to those
presented in Fig. 4 were performed. The
ability of ssDNA and dsDNA to inhibit the unwinding reaction was
tested, and in contrast to ssRNA and dsRNA, neither ssDNA nor dsDNA
caused inhibition of eIF4A unwinding activity when titrated into the
reaction (data not shown and Fig. 4B). Similarly, an
ssDNA-PS oligonucleotide was not an effective inhibitor of unwinding
(<15%, 1.0 µM ssDNA-PS) when tested in the competitive
inhibition assay (data not shown). To investigate the possibility that
eIF4A was able to bind, but not unwind an RNA/2'-MOE duplex, both a
single-stranded 2'-MOE and a blunt-ended RNA/2'-MOE duplex
(R-13C/2'-MOE-13) were tested in the competition assay. Results
indicated that neither was effective at inhibiting the helicase
reaction (up to 1.0 µM was tested, data not shown). Taken
together, these results demonstrate that under the assay conditions
used, the relative affinity of eIF4A for ssDNA, dsDNA, single-stranded
2'-MOE, or RNA/2'-MOE duplexes is much weaker than for ssRNA or
dsRNA.
That single-stranded DNA did not act as a competitive inhibitor
was surprising, since data had shown that eIF4A does bind to DNA when
measured via the RNA-dependent ATPase activity of eIF4A
(37). Therefore, this inconsistency was investigated further. Comparing
reaction conditions between those used here for the unwinding activity
and those used by Peck and Herschlag (37) for the
RNA-dependent ATPase activity showed major differences in
the pH, ion type, and ionic strength. For the helicase reaction, the
typical buffer contains 20 mM HEPES-KOH, pH 7.2, and 70 mM potassium chloride (Buffer A), whereas for the ATPase
reaction, the typical buffer contains 25 mM MES-KOH, pH
6.0, and 10-30 mM potassium acetate (Buffer B). Therefore,
similar competitive inhibition studies to those above were performed
under Buffer B conditions. It should be noted that due to the buffer in
which reticulocyte eIF4A is purified and stored, it was possible to
only decrease the total potassium chloride concentration in the
reaction to 15 mM, and thus a final concentration of 15 mM potassium acetate was used to give a combined monovalent
ionic strength of 30 mM (Buffer B'). Under these Buffer B'
conditions, the unwinding activity of eIF4A in the absence of any
potential inhibitor was ~80% of the unwinding activity observed
under Buffer A conditions (data not shown). When the ssRNA or ssDNA
competitor was added to a final concentration of 1.0 µM,
the unwinding activity of eIF4A was reduced to ~25 and 70%,
respectively, of the total unwinding activity observed under Buffer B'
conditions (data not shown). Thus, limited interaction of eIF4A with
ssDNA is observed when the pH and ionic strength of the buffer are
altered from conditions A to conditions B'. These results indicate that
altering the buffer conditions can result in differences in the ability
of eIF4A to interact with single-stranded and double-stranded nucleic
acids, as monitored by inhibition of the unwinding reaction.
Substrate Specificity of eIF4A--
Previous work (8) demonstrated
that the initial rate and amplitude of duplex unwinding by eIF4A was a
function of duplex stability. This relationship has been more
rigorously investigated by using a series of RNA duplex substrates in
which the effects of duplex length and duplex stability on the degree
of unwinding have been tested independently of each other. This study
confirms that the degree of duplex unwinding by eIF4A is dependent only on the overall stability (
Investigation of the dependence of unwinding on the direction and
length of the single-stranded region of duplex substrates indicated
that the efficiency of eIF4A helicase activity is approximately equal
for the 5'
Although the data support the idea that eIF4A unwinds a blunt-ended
duplex by directly interacting with the double-stranded region, it
cannot be explicitly ruled out that eIF4A is recognizing short
single-stranded regions at the ends of the duplex due to "breathing" or "fraying" of the terminal base pair(s). However, if breathing was a major feature of the ability of eIF4A to
unwind blunt-ended duplexes by presenting short single-stranded
regions, then one would expect to observe a much greater increase in
the degree of unwinding as the single-stranded region adjacent to the
duplex was increased from 0 to 4 nts. Furthermore, the potential for
breathing in the 13-bp blunt-ended duplex is decreased as 2 of the 3 bp
at both ends of the duplex are G:C. In addition, it has been
demonstrated that the site size for optimal binding and ATPase activity
by eIF4A is 13-15 nt (37, 49).
Experiments designed to test the effect of chemical modifications of
the duplex on helicase activity show that eIF4A is able to unwind
RNA/RNA, RNA/DNA, and DNA/RNA but not DNA/DNA duplexes (Fig.
5 and Table II). In addition, the
stability of the duplex, regardless of composition (RNA/RNA, RNA/DNA,
or DNA/RNA) or length, dictates how efficiently eIF4A will unwind a
particular duplex (Table II). Results indicated that although a change
from a PO to a PS backbone had no effect on eIF4A helicase activity,
substrates containing a 2'-MOE modification abrogated unwinding
activity by eIF4A (Fig. 5 and Table II).
Taken together, the results of this study provide additional detail
with respect to how eIF4A interacts with and unwinds duplex substrates.
First, eIF4A is not limited to interacting with only ssRNA but also may
interact directly with dsRNA. Second, it is evident that an RNA strand
(2'-OH) is required for eIF4A helicase activity. This agrees with the
finding that 2'-OH groups are necessary for stimulating eIF4A ATPase
activity (37, 49). Third, eIF4A may bind the RNA strand in either
orientation (5'
The data presented here suggest that eIF4A may recognize and bind
to only one strand (which must be RNA) of double-stranded nucleic acids
and that any interaction with the complementary strand is minimal. This
is supported by the results that RNA/RNA, RNA/DNA, RNA/DNA-PS, and
DNA/RNA but not DNA/DNA duplexes are unwound by eIF4A and that ssRNA
and dsRNA serve as competitive inhibitors of the helicase reaction,
whereas ssDNA, ssDNA-PS, and dsDNA do not serve as competitive
inhibitors. Thus, the recognition of single-stranded and
double-stranded nucleic acids (as helicase substrates) by eIF4A is
based on the presence of a 2'-OH ribose moiety. The possibility that
eIF4A interacts with only one (RNA) strand of a double-stranded nucleic
acid is similar to recent observations (55) made with the HCV NS3
helicase using a series of RNA/RNA, RNA-Me/RNA-Me and RNA/RNA-Me hybrid
duplexes. This is also similar to the RecBC helicase, which interacts
and translocates along only one strand (the 3'
Whereas our results suggest that eIF4A may interact with only one
strand of a nucleic acid duplex and that this strand must be RNA
(2'-OH), it cannot be explicitly ruled out that the differences with
which eIF4A interacts with dsRNA and dsDNA are based on structural, rather than chemical, features of double-stranded nucleic acids. That
is, eIF4A is able to recognize A-form helices (dsRNA), but not B-form
helices (dsDNA), and implies that eIF4A would interact with both
strands of the duplex substrate. Structural studies of RNA/DNA (or
DNA/RNA) duplexes indicate that while these molecules have an A-form
character, the RNA and DNA chains in these hybrid duplexes adopt
different helical conformations (57). The RNA strand retains the A-form
helical conformation, whereas the DNA strand adopts a conformation that
is neither A-form nor B-form but is intermediate in character between
these two forms (57). This may explain how eIF4A recognizes and unwinds
RNA/DNA and DNA/RNA duplexes. However, whereas the structures of short
ssRNA and ssDNA oligonucleotides differ in their degree of rigidity, neither adopts a "standard" structural form like dsRNA and dsDNA. Thus, the ability of eIF4A to interact with (or unwind) ssRNA, dsRNA,
RNA/DNA, RNA/DNA-PS, and DNA/RNA but not ssDNA, ssDNA-PS, or dsDNA may
be due to differences in either the chemical composition of the nucleic
acids (2'-OH versus 2'-H) or helical structure (A-
versus B-form helices). It is also quite possible that
both the chemical composition and physical characteristics
of single-stranded and double-stranded nucleic acids play a role in
eIF4A substrate recognition.
A Revised Model for Duplex Unwinding by eIF4A--
Previous
results obtained in this laboratory led to a simple model by which
eIF4A unwinds duplexes in an ATP-dependent manner (see
Introduction and Ref. 8). The results obtained in this report support
the basic tenets of this model and offer more details concerning the
mechanism of unwinding by eIF4A. Our previous model suggested that
eIF4A must bind to a ssRNA region before translocation and duplex
unwinding occurs. However, the results presented in this report suggest
that eIF4A is capable of binding to the double-stranded region of a
partial or fully duplexed nucleic acid substrate. Therefore, a revised
model of how eIF4A initiates unwinding is presented in Fig.
6. This model shows that unwinding may
initiate from two possible pathways. In the first pathway (1),
eIF4A·ATP binds to the single-stranded region of the substrate and
translocates to the double-stranded region, where it initiates
unwinding and displaces the release strand. Note that this pathway is
identical to that proposed previously; however, it is unclear if more
than one ATP hydrolysis event is needed for translocation
and unwinding. Since the binding of eIF4A to ssRNA is weak
(Kd
In the second pathway (2), eIF4A·ATP binds directly to and
initiates unwinding at the duplex region. Note that this pathway eliminates the necessity for a translocation event and that a single
ATPase event may be sufficient for strand separation. It is unclear,
however, whether eIF4A initiates unwinding at the end of the duplex
(Pathway 2A) or at an internal region of the duplex (Pathway 2B).
Pathway 2 is likely the only means by which eIF4A could unwind
blunt-ended and DNA/RNA duplexes. Note that in both pathways, the
ability of eIF4A to unwind the duplex completely is still related to
the overall stability of the substrate, and recognition of the duplex
may be due to chemical and/or structural features of the substrate.
eIF4A: Nucleic Acid Binding, ATPase Activity, and Helicase
Activity--
Comparison of the results presented in this report with
previous studies (35-37, 49) show a number of inconsistencies among the nucleic acid binding, ATP hydrolysis, and helicase activities of
eIF4A. These differences focus on the chemical and physical properties
of various nucleic acids that will (or will not) serve as substrates
for binding, stimulating ATP hydrolysis and helicase activity. Many of
these inconsistencies may be explained by the fact that the binding
ATPase and helicase experiments were performed under markedly different
reaction conditions, including buffer composition, protein and
substrate concentration, and source of eIF4A (8, 35-37, 49). This is
strongly supported by a series of experiments demonstrating that
coupling between the binding of ligands (ssRNA and ATP) and the number
of conformational states available to eIF4A are markedly different
under various salt and pH conditions (36).
These apparent differences between the ATPase and helicase activities
of eIF4A imply that the relationship between these two enzymatic
activities may be much more complex than originally predicted. In
general, it appears that the large stimulation of eIF4A ATPase activity
observed in the presence of ssRNA may not be absolutely required for
efficient strand separation. This may reflect differences in the
microscopic rate constants and/or rate-limiting steps between the
ATPase and helicase reactions. A similar disparity between the ATPase
and helicase activities of the HCV NS3 helicase has recently been
investigated in detail, and it was concluded that the binding of ssRNA
by HCV NS3 necessary for maximal stimulation of ATPase activity is not
directly related to nucleic acid duplex unwinding (55).
In conclusion, this report has further characterized the helicase
activity of eIF4A with respect to substrate specificity and provides
additional insight into the mechanism of duplex unwinding by
DEAD/DEXH box proteins in general. Further investigation
will be required to fully understand the complex relationship between the nucleic acid binding, ATP hydrolysis, and helicase activities of
this important class of proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and [
-32P]CTP from
PerkinElmer Life Sciences; DNA oligonucleotides PTT-23, PTB-44, and
D-14, D-15, D-16, D-17, D-20, and D-20C from the Molecular Biology Core
Laboratory, Case Western Reserve University; DNA oligonucleotides
PTB-41SD, PTB-41S-, PTB-41S+, PTB-44L+, and D-44 from IDT,
Inc., Coralville, IA; RNA oligonucleotides R-10, R-11, R-12, R-13,
R-14, and R-15 from Cybersyn, Lenni, PA; RNA oligonucleotides R-17-5',
R-19-5', R-21-5', R-23-5', R-28-5', R-38-5', R-17-3', R-19-3',
R-21-3', R-23-3', R-28-3', R-38-3' R-17C, R-13C, R-13SD, R-13S-,
R-13S+, and R-16L+ from Dharmacon, Inc, Boulder, CO;
MegashortscriptTM In Vitro transcription kit
from Ambion; and T4 polynucleotide kinase from New England Biolabs.
DNA-PS, 2'-MOE, and 2'-MOE/PS oligonucleotides were supplied by Isis
Pharmaceuticals, Carlsbad, CA.
-32P]CTP
(specific activity 3000 Ci/mmol, 10 mCi/ml) as a tracer label. Products
of the reactions were purified on a denaturing (7 M urea)
14% polyacrylamide (19:1 acrylamide:bisacrylamide) gel. The proper
bands were located by autoradiography, excised from the gel, and the
RNA eluted from the gel slices in 400 µl of 0.5 M sodium
acetate, pH 5.2, 1 mM EDTA, and 3% phenol:chloroform (v/v)
for 3-4 h at 4 °C. The gel slices were removed from the solution by
centrifugation through quick-sep columns, and the recovered RNAs
were phenol:chloroform-extracted once and ether-extracted twice,
followed by ethanol (1 ml) precipitation for 12-24 h at
20 °C.
The precipitated RNAs were recovered by centrifugation at 14,000 rpm at
4 °C for 60 min. The supernatant was removed, and the RNA pellets
washed in ice-cold 70% ethanol, dried, and resuspended in
distilled H2O. Quantitation of each purified
transcript was performed by UV spectroscopy as described above.
-32P]ATP (specific activity 3000 Ci/mmol, 10 mCi/ml) were combined with 10 units of T4 polynucleotide
kinase (10 µl final volume), and reactions were performed as
described (41). Percent incorporation of the label into these
oligonucleotides is 90-95%, and specific activities of the
oligonucleotides are on the order of 1 × 106
cpm/pmol. Forty picomoles of DNA-PS, 2'-MOE, or 2'-MOE/PS
oligonucleotides (see Table I) and 100 pmol of
[
-32P]ATP (specific activity 6000 Ci/mmol, 150 mCi/ml)
were combined with 10 units of T4 polynucleotide kinase (10 µl final
volume) and reactions were performed as described above. Percent
incorporation of the label into these oligonucleotides is 1-5%, and
specific activities of the oligonucleotides are on the order of
5×105 cpm/pmol.
20 °C and diluted in 1×
hybridization buffer for use in the helicase assay.
80 °C for using intensifying screens.
) eIF4A was incubated at
4 °C during the course of each experiment, and the amount of duplex
in this control reaction (typically 36-38 fmol) was normalized to
100%, and the amount of (background) single-stranded RNA (typically
2-4 fmol) was normalized to 0%. All other reactions were scaled to
these values. For the competition experiments (Fig. 4), the percentage
of inhibition is given by 100
((% unwinding of R-41S-/R-13S-
in the presence of x µM inhibitor)/(%
unwinding of R-41S-/R-13S- in the presence of 0.0 µM
inhibitor) × 100).
G) values for RNA/RNA
duplex substrates were calculated for 35 °C using the nearest
neighbor method of analysis as described by Turner and Sugimoto (42).
Duplex stability (
G) values for RNA/DNA and DNA/RNA
duplex substrates were calculated for 35 °C using the nearest
neighbor method of analysis as described by Sugimoto et al.
(43). Duplex stability (
G) values for DNA/DNA duplex
substrates were calculated for 35 °C using the nearest neighbor
method of analysis as described by SantaLucia et al. (44).
Changing the nonbridging oxygen atom in the oligonucleotide phosphodiester (PO) backbone to sulfur (phosphorothioate (PS) backbone)
decreases the stability of the duplex by 0.3 kcal/mol per modification
(45). Changing the 2'-group of the ribose sugar to a methoxyethyl (MOE)
group increases the stability of the duplex by 0.2 kcal/mol per
modification (46-48).
G value) for duplexes with
stabilities between
17.9 kcal/mol and
24.7 kcal/mol. (Fig.
1C).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G value) increased (8). Furthermore, it was observed
that a linear correlation exists between the ln(initial rate of
unwinding) and
G value of the duplex. It was demonstrated that eIF4A was able to efficiently unwind an 11-base pair (bp) duplex
having a stability of
17.9 kcal/mol (8); however, former studies
showed that eIF4A was unable to unwind a 10-bp duplex with a stability
of approximately
25.0 kcal/mol (38). Taken together, this suggests
that the amount of unwinding by eIF4A is dependent only on the
stability, rather than the length, of the duplex substrate. Thus, we
wished to test more rigorously the effects of duplex length and
stability on eIF4A helicase activity. In addition, it was previously
observed that the maximum percentage of duplex unwound by eIF4A also
decreased as the stability of the duplex increased (8). Therefore, we
also further investigated the relationship between the maximum
percentage of duplex unwinding (amplitude) by eIF4A and duplex stability.
G =
17.9,
20.6, and
23.3
kcal/mol) were designed and tested in the helicase assay. Fig.
1A shows typical experimental
data for the unwinding of RNA duplexes by eIF4A. It is apparent from
Fig. 1A that the degree of unwinding by eIF4A decreases as
the stability of the duplex is increased. In addition, a 16-bp duplex
(
G =
20.5 kcal/mol) was unwound to approximately
the same degree as the 13-bp duplex of the same stability
(
G =
20.6 kcal/mol). These results show that the
amplitude of duplex unwinding decreases as the stability, but not the
length, of the duplex is increased. Furthermore, it shows that two
duplexes of different length but of equal stability are unwound by
eIF4A with similar efficiencies. These results indicate that duplex stability, rather than length, is the primary determinant for how
efficiently eIF4A is able to unwind an RNA duplex.
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Fig. 1.
The degree of unwinding duplexes by eIF4A is
dependent on the stability (rather than the length) of the duplex for
short RNA duplex substrates. A, autoradiograms
illustrating the degree of unwinding three 13-bp duplexes of differing
stabilities and one 16-bp duplex by eIF4A (R-41S-/R-13S-,
G =
17.9 kcal/mol; R-41SD/R-13SD,
G =
20.6 kcal/mol; R-41S+/R-13S+,
G =
23.3 kcal/mol; R-44L+/R-16L+,
G =
20.5 kcal/mol). Lane designated as
protein was incubated at 4 °C.
denotes
that the duplex was heated to 95 °C for 5 min. Lane designated as
protein, 35 °C demonstrates that minimal duplex
unwinding (thermal melting) occurs in the absence of eIF4A (15 min).
Note that the 1st three lanes (
protein;
;
protein, 35 °C) show representative controls
for the 13-bp,
17.9 kcal/mol duplex (R-41S-/R-13S-); controls for the
other duplex substrates used showed similar results. Lanes designated
as +eIF4A indicate the duplex was incubated with 0.8 µM
eIF4A for 15 min (representing maximal unwinding) with the indicated
duplex substrate. B, plot illustrating the linear
relationship between ln(initial rate of unwinding) (
) or ln(initial
rate of thermal melting) (
) and duplex stability for the R-44/R-10,
-11, -12, -13, -14, and -15 duplexes. C, plot illustrating
the linear relationship between the ln(amplitude of unwinding)
versus duplex stability for the R-44/R-11, -12, -13, and -14 duplexes (
) and the R-41S-/R-13S-, R-41SD/R-13SD, R-41S+/R-13S+, and
R-44L+/R-16L+ duplexes (
). The ln(amplitude of thermal melting)
versus duplex stability for the R-44/R-10, -11, -12, and -13 duplexes is designated by (
). Note that the values of the slopes in
B and C are equivalent, suggesting that the
relationship between the ln(amplitude of unwinding) and duplex
stability is similar to the relationship between the ln(initial rate of
unwinding) and duplex stability. Error bars are omitted for
clarity; the standard error is less than ±5% for all data points in
this series of experiments.
0.24). It
should be noted that although the length and nucleotide sequences of
the duplexes tested in Fig. 1A are different from the R-44
series of duplexes tested (Table I), the
ln(amplitude of eIF4A-dependent unwinding) for the two sets
of points may be fitted to a single line.
Characteristics of DNA and RNA oligonucleotides
3' and 3'
5' directions (38). Thus, it is unknown if
this bidirectional activity is a function inherent to eIF4A or if eIF4B
is necessary for bidirectional unwinding.
3' and 3'
5' substrates (Table II).
Presented in Fig. 2B are experimental data of eIF4A
unwinding duplexes with 0- (blunt-ended) and 25-nt 5'-single-stranded
regions, and the complete set of data is summarized graphically in Fig.
2C. These data indicate that eIF4A alone is capable of
bidirectional unwinding activity, and the degree of unwinding is
approximately equal in both directions (i.e. the lines in
Fig. 2C are similar), although eIF4A appears to be slightly more active in unwinding in a 5'
3' direction at shorter (0-8 nt)
single-stranded region lengths. It can also be seen from Fig. 2C that there is little, if any, change in the degree of
unwinding as the length of the single-stranded region is increased
beyond 8 nt for either 5'
3' or 3'
5' unwinding. In addition,
eIF4A unwinding activity decreases only slightly as the length of
single-stranded region is shortened below 8 nt. Most surprising was the
observation that eIF4A is capable of unwinding a duplex with neither
5'- nor 3'-single-stranded regions (blunt-ended) with ~70% of the
activity observed for duplexes with at least 8-nt single-stranded
regions.
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Fig. 2.
Effect of the length and directionality of
the ssRNA region on the duplex unwinding activity of eIF4A.
A, schematic representation of the 5'- and 3'-ssRNA region
duplexes used in this series of experiments. B, selected
autoradiograms illustrating the unwinding of RNA duplexes with 0- and
25-nt 5'-ssRNA regions. Lanes designated as protein were
incubated at 4 °C.
denotes that the duplex was heated
to 95 °C for 5 min. Lanes designated as
protein,
35 °C demonstrate that minimal duplex unwinding (thermal
melting) occurs in the absence of eIF4A (15 min). Lanes designated as
eIF4A indicate the duplex was incubated with 0.8 µM eIF4A for 15 min. C, plot of eIF4A duplex
unwinding activity (amplitude) with respect to the length of the 5'-
(
) and 3'- (
) single-stranded region. The duplexes used are as
follows: R-38, -28, -23, -21, -19, -17-5'/R-13; R-13/R-13C; R-38, -28, -23, -21, -19, -17-3'/R-13C.
Characteristics of duplex substrates and maximum percent unwound by
eIF4A
30%) when compared with the
efficiency of unwinding a duplex substrate containing a single-stranded
region adjacent to the duplex region.
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Fig. 3.
Effect of eIF4A concentration on the
unwinding of a blunt-ended duplex. Titration of eIF4A into the
helicase reaction using the R-38-5'/R-13 duplex containing a 25-nt
single-stranded region ( ) and the R-13/R-13C (blunt-ended) duplex
(
). Note that the amplitude of unwinding the R-13/R-13C duplex is
consistently 70% of the amplitude of unwinding the R-38-5'/R-13
duplex for all eIF4A concentrations tested.
PS) and 2'-substituent (2'-H
2'-MOE) modifications used. Note
that changing PO
PS decreases the stability of the duplex by 0.3 kcal/mol per modification, and substituting MOE for the 2'-OH increases
the stability of the duplex by 0.2 kcal/mol per modification (45-48).
The unwinding assay was performed with the modified substrates, and the
RNA/RNA and RNA/DNA duplexes were used in parallel reactions for
comparison. Fig. 5A presents selected raw data for unwinding
RNA/DNA-PS, RNA/2'-MOE, and RNA/2'-MOE/PS duplexes, and the amplitudes
of unwinding for these modified duplexes by eIF4A are listed in Table
II. The PS modification appeared to have no effect on the helicase
activity of eIF4A, as the magnitude of unwinding the R-44/D-PS-16
duplex was in the expected range for its stability (compare with the
stabilities and amplitudes of unwinding the R-44/R-11 and R-44/R-12
duplexes, Table II). In contrast, the amplitudes of unwinding the
duplex substrates containing either 2'-MOE or 2'-MOE/PS modifications
were much less than predicted by their relative stabilities (compare
R-44/2'-MOE-11,
G
16.7 kcal/mol, amplitude
10% with R-44/R-11,
G
17.9 kcal/mol,
amplitude
70%, etc.) In fact, all of the duplexes with 2'-MOE or
2'-MOE/PS modifications were unwound to
10% or less (Fig.
5A and Table II). Taken together, these results demonstrate that PO
PS modifications of the DNA strand in an RNA/DNA duplex have little or no effect on unwinding activity, whereas a 2'-H
2'-MOE modification of one strand in a duplex abrogates unwinding by eIF4A.
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Fig. 4.
Helicase activity of eIF4A is inhibited by
excess ssRNA or dsRNA but not ssDNA or dsDNA. A,
autoradiogram illustrating that titration of dsRNA competitor into the
helicase reaction inhibits eIF4A duplex unwinding activity. Lane
designated as protein was incubated at 4 °C.
denotes that the duplex was heated to 95 °C for 5 min. Lanes designated as +eIF4A, +µM
dsRNA indicate that 2.0 nM duplex substrate was
combined with 0.8 µM eIF4A, and then dsRNA competitor was
added to give the final µM concentration indicated
above each lane, followed by incubation at 35 °C for 15 min. B, plot of the normalized % inhibition with respect to
increasing competitor concentration. 100% duplex unwound is defined as
the amount of unwinding in 15 min for the R-41S-/R-13S- substrate in
the absence (0 µM) of ssRNA (ssDNA) or dsRNA (dsDNA)
competitor, respectively. The amount of unwinding in reactions
containing competitors are normalized to this value and then subtracted
from 100% to give the percentage inhibition value shown in
B (see "Experimental Procedures").
, ssRNA
competitor, R-17C;
, dsRNA competitor, R-17-3'/R-17C;
, ssDNA
competitor, D-20;
, dsDNA competitor, D-20/D-20C. Error
bars are omitted for clarity; the standard error is less than
±5% for all data points in this series of experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G value) of the duplex and is
independent of duplex length or nucleotide sequence (Fig. 1,
A and C). In addition, current results
demonstrate that like the initial rate, the ln(amplitude of
eIF4A-dependent unwinding or thermal melting) is also
linearly correlated with the stability of the duplex (Fig. 1,
B and C), and the values of these slopes are
equivalent to those of the ln(initial rate of unwinding or thermal
melting) versus duplex stability. This indicates that the
amplitude of unwinding may also be used to compare the efficiency of
eIF4A-dependent unwinding among different types of duplex
substrates. It is stressed that the unwinding assay used here allows
the measurement of only fully unwound duplexes. Thus, only the overall
macroscopic rate (or amplitude) of the unwinding process may be
observed, and the many potential microscopic kinetic steps of the
unwinding reaction (including but not limited to: ATP and duplex
binding, ATP hydrolysis, duplex unwinding, release of products, duplex
reannealing, and "snap-back") are unable to be characterized in
detail. Although limited, this assay does allow for the comparison of
eIF4A helicase activity with respect to differences in duplex
stability, chemical composition, or physical characteristics and,
combined with previous RNA binding and ATP hydrolysis data, allows
simple models of the duplex unwinding mechanism to be proposed (see below).
3' and 3'
5'directions and that bidirectional unwinding is a property of eIF4A and is not dependent on the presence of other initiation factors (Fig. 2). This bidirectional helicase activity supports the idea that eIF4A is involved in melting short RNA
duplex structures in mRNA, rather than acting as a processive, directional helicase. Furthermore, the unwinding activity of eIF4A is
minimally dependent on the length of the single-stranded region adjacent to the duplex region of the substrate. Most interesting is the
fact that eIF4A is able to unwind a blunt-ended duplex with only a
modest (30%) decrease in activity when compared with duplexes with
4-25-nt single-stranded regions (Figs. 2 and 3). This suggests that a
single-stranded region preceding the duplex is not a requirement for
eIF4A-dependent duplex unwinding and that eIF4A may
interact directly with dsRNA. This is supported by the competition
experiments showing that like ssRNA, dsRNA acts as an inhibitor of
eIF4A helicase activity (Fig. 4). Taken together, this suggests that
eIF4A also binds to dsRNA but with a decrease in relative affinity when
compared with ssRNA. The ability of eIF4A to unwind blunt-ended
duplexes in vitro may indicate its being able to disentangle
highly structured and/or sequestered duplex region in the
5'-untranslated region of mRNAs.
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Fig. 5.
Effect 2'-ribose group and backbone
modifications on the duplex unwinding activity of eIF4A.
A, selected autoradiograms illustrating the unwinding of
RNA/RNA, RNA/DNA, DNA/RNA, DNA/DNA, RNA/DNA-PS, RNA/2'-MOE, and
RNA/2'-MOE/PS duplexes by eIF4A. Lane designated as
protein was incubated at 4 °C.
denotes
that the duplex was heated to 95 °C for 5 min. Lane designated as
protein, 35 °C demonstrates that minimal duplex
unwinding (thermal melting) occurs in the absence of eIF4A (15 min).
Lanes designated as +eIF4A indicate the duplex was incubated
with 0.8 µM eIF4A for 15 min. Note that the 1st
three lanes (
protein;
;
protein,
35 °C) show representative controls for the 12 bp,
21.4
kcal/mol duplex (R-44/R-12); controls for the other duplex substrates
used showed similar results. See Table II for all duplexes used.
B, diagrams of the PS backbone (46) and 2'-MOE
modifications.
3' or 3'
5') since duplex substrates with 5'
and 3' single-stranded regions are unwound with similar efficiencies by
eIF4A. This is further supported by the fact that both RNA/DNA and
DNA/RNA duplex are unwound, since the orientation of the RNA strand
relative to the DNA strand is opposite for each duplex. Finally, the
molecular basis for the inability of eIF4A to unwind an RNA/2'-MOE or
RNA/2'-MOE/PS duplex is likely due to the large 2'-MOE group
obstructing the binding interface between eIF4A and the duplex. In
support of this hypothesis, it has been shown via NMR structural
studies that the MOE group occupies the minor groove of an RNA/2'-MOE duplex (53, 54) and suggests that the contacts made between eIF4A and
double-stranded nucleic acids may involve the minor groove of the duplex.
5' strand) of DNA
duplex substrates (56).
100 µM, (35, 49, 58)), it is
possible that eIF4A undergoes several rounds of ATP hydrolysis,
dissociation, and reassociation to the substrate before interacting
with the double-stranded region of the molecule. Thus, the presence of
the single-stranded region may help to build a higher local
concentration of eIF4A·ATP near the double-stranded region of the
substrate, and thus "translocation" is actually a
dissociation/reassociation event as opposed to continuous movement
along the ssRNA. This may explain why the degree of
eIF4A-dependent unwinding is slightly higher with
substrates containing an ssRNA region when compared with blunt-ended
substrates.
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Fig. 6.
Revised model of eIF4A-dependent
RNA duplex unwinding. Schematic diagram illustrating possible
pathways by which eIF4A unwinds RNA duplex substrates. In this model,
eIF4A is depicted as a two domains (N-terminal, trapezoid;
C-terminal, oval) connected by a distended polypeptide
linker (curved line) in accordance with the structural
results obtained by Caruthers and McKay (33). Both pathways begin with
eIF4A binding to ATP ( ). Pathway 1 shows eIF4A binding
the ssRNA region of a duplex substrate. In this pathway, eIF4A must
translocate to the dsRNA region, which may require ATP hydrolysis and
nucleotide exchange. eIF4A, now positioned at the duplex region (equal
to n base pairs), hydrolyzes ATP to ADP (
) and unwinding
initiates at the ss/ds junction. A small number of base pairs
(e.g. 4) are unwound, and if enough destabilization occurs,
the strands separate from each other. This is followed by complex
(eIF4A·ADP·RNA) dissociation. Pathway 2 shows
eIF4A binding to a blunt-ended duplex. Note that eIF4A is positioned
directly at the double-stranded region, and no translocation occurs.
eIF4A hydrolyzes ATP, and unwinding initiates at either the end of the
duplex (pathway 2A) or at an internal region of the duplex
(pathway 2B). A small number of base pairs (e.g.
4) are unwound, and if enough destabilization occurs, the strands
separate from each other. This is followed by complex
(eIF4A·ADP·RNA) dissociation. (i) The degree of strand separation
(as opposed to reannealing or snap-back) is dependent on duplex
stability. (ii) It is possible that eIF4A may bind directly to the
double-stranded region of a substrate containing both and
single-stranded and double-stranded regions and proceed via
pathway 2A or 2B. See text for additional
details.
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ACKNOWLEDGEMENTS |
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We thank Dr. David R. Setzer for the use of the Ambis radioanalytical scanner to quantitate the helicase experiments. We also thank Dr. Pieter de Haseth, Dr. Vernon Anderson, Dr. Jack Hensold, and Tracy Mourton for their advice and discussion.
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Note Added in Proof |
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Recently the crystal structure of full-length elF4A has been published (Caruthers, J. M., Johnson, E. R., and McKay, D. B. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13080-13085).
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Research Grant GM26796 (to W. C. M.) and by Research Training Grant in Metabolism DK07319 (to G. W. R.).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.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4935. Tel.: 216-368-3578; Fax: 216-368-3419; E-mail: wcm2@po.cwru.edu.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M007560200
2 Thermal melting is defined as any strand separation of duplex nucleic acid that occurs as a passive thermal process; the term unwinding is reserved for strand separation of duplex nucleic acid that occurs in an eIF4A-dependent manner.
3 RNA/DNA refers to a duplex composed of a long strand of RNA and a short strand of DNA. Conversely, DNA/RNA refers to a duplex composed of a long strand of DNA and a short strand of RNA.
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ABBREVIATIONS |
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The abbreviations used are: Met-tRNAi, initiator methionyl-tRNA; eIF, eukaryotic initiation factor; CWRU MBCL, Case Western Reserve University Molecular Biology Core Laboratory; PO, phosphate; PS, phosphorothioate; MOE, methoxyethyl; BSA, bovine serum albumin; MES, 2-(N-morpholino)ethanesulfonic acid; ssRNA, single-stranded RNA; dsRNA, double-stranded RNA; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; bp, base pair(s); nt, nucleotide(s).
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Merrick, W. C., and Hershey, J. W. B. (1996) in Translational Control (Matthews, M. B. , Sonenberg, N. , and Hershey, J. W. B., eds) , pp. 31-69, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
2. | Kozak, M. (1999) Gene 234, 187-208[CrossRef][Medline] [Order article via Infotrieve] |
3. | Pain, V. M. (1996) Eur. J. Biochem. 236, 747-771[Abstract] |
4. |
Richter, N. J.,
Rogers, G. W., Jr.,
Hensold, J. O.,
and Merrick, W. C.
(1999)
J. Biol. Chem.
274,
35415-35424 |
5. | Linder, P., Lasko, P. F., Ashburner, M., Leroy, P., Nielsen, P. J., Nishi, K., Schnier, J., and Slonimski, P. P. (1989) Nature 337, 121-122[CrossRef][Medline] [Order article via Infotrieve] |
6. | de la Cruz, J., Kressler, D., and Linder, P. (1999) Trends Biochem. Sci. 24, 192-198[CrossRef][Medline] [Order article via Infotrieve] |
7. | Luking, A., Stahl, U., and Schmidt, U. (1998) Crit. Rev. Biochem. Mol. Biol. 33, 259-296[Abstract] |
8. |
Rogers, G. W., Jr.,
Richter, N. J.,
and Merrick, W. C.
(1999)
J. Biol. Chem.
274,
12236-12244 |
9. |
Iost, I.,
Dreyfus, M.,
and Linder, P.
(1999)
J. Biol. Chem.
274,
17677-17683 |
10. | Wang, Y., Wagner, J. D., and Guthrie, C. (1998) Curr. Biol. 8, 441-451[Medline] [Order article via Infotrieve] |
11. |
Wagner, J. D.,
Jankowsky, E.,
Company, M.,
Pyle, A. M.,
and Abelson, J. N.
(1998)
EMBO J.
17,
2926-2937 |
12. |
Okanami, M.,
Meshi, T.,
and Iwabuchi, M.
(1998)
Nucleic Acids Res.
26,
2638-2643 |
13. | Valdez, B. C., Henning, D., Perumal, K., and Busch, H. (1997) Eur. J. Biochem. 250, 800-807[Abstract] |
14. | Burgess, S. M., and Guthrie, C. (1993) Trends Biochem. Sci. 18, 381-384[CrossRef][Medline] [Order article via Infotrieve] |
15. | Staley, J. P., and Guthrie, C. (1998) Cell 92, 315-326[Medline] [Order article via Infotrieve] |
16. | Tai, C. L., Chi, W. K., Chen, D. S., and Hwang, L. H. (1996) J. Virol. 70, 8477-8484[Abstract] |
17. | Gross, C. H., and Shuman, S. (1995) J. Virol. 69, 4727-4736[Abstract] |
18. |
Gwack, Y.,
Yoo, H.,
Song, I.,
Choe, J.,
and Han, J. H.
(1999)
J. Virol.
73,
2909-2915 |
19. | Zhong, W., Ingravallo, P., Wright-Minogue, J., Skelton, A., Uss, A. S., Chase, R., Yao, N., Lau, J. Y., and Hong, Z. (1999) Virology 261, 216-226[CrossRef][Medline] [Order article via Infotrieve] |
20. | Warrener, P., and Collett, M. S. (1995) J. Virol. 69, 1720-1726[Abstract] |
21. | Stauber, N., Martinez-Costas, J., Sutton, G., Monastyrskaya, K., and Roy, P. (1997) J. Virol. 71, 7220-7226[Abstract] |
22. |
Bisaillon, M.,
Bergeron, J.,
and Lemay, G.
(1997)
J. Biol. Chem.
272,
18298-18303 |
23. | Jankowsky, E., Gross, C. H., Shuman, S., and Pyle, A. M. (2000) Nature 403, 447-451[CrossRef][Medline] [Order article via Infotrieve] |
24. | Schmid, S. R., and Linder, P. (1992) Mol. Microbiol. 6, 283-291[Medline] [Order article via Infotrieve] |
25. | Pause, A., and Sonenberg, N. (1992) EMBO J. 11, 2643-2654[Abstract] |
26. | Pause, A., Methot, N., and Sonenberg, N. (1993) Mol. Cell. Biol. 13, 6789-6798[Abstract] |
27. | Gross, C. H., and Shuman, S. (1996) J. Virol. 70, 1706-1713[Abstract] |
28. | Kim, D. W., Kim, J., Gwack, Y., Han, J. H., and Choe, J. (1997) J. Virol. 71, 9400-9409[Abstract] |
29. | Wardell, A. D., Errington, W., Ciaramella, G., Merson, J., and McGarvey, M. J. (1999) J. Gen. Virol. 80, 701-709[Abstract] |
30. |
Lin, C.,
and Kim, J. L.
(1999)
J. Virol.
73,
8798-8807 |
31. | Benz, J., Trachsel, H., and Baumann, U. (1999) Struct. Fold. Des. 15, 671-679 |
32. |
Johnson, E. R.,
and McKay, D. B.
(1999)
RNA
5,
1526-1534 |
33. |
Caruthers, J. M.,
Johnson, E. R.,
and McKay, D. B.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
13080-13085 |
34. | Bird, L. E., Subramanya, H. S., and Wigley, D. B. (1998) Curr. Opin. Struct. Biol. 8, 14-18[CrossRef][Medline] [Order article via Infotrieve] |
35. | Lorsch, J. R., and Herschlag, D. (1998) Biochemistry 37, 2180-2193[CrossRef][Medline] [Order article via Infotrieve] |
36. | Lorsch, J. R., and Herschlag, D. (1998) Biochemistry 37, 2194-2206[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Peck, M. L.,
and Herschlag, D.
(1999)
RNA
5,
1210-1221 |
38. | Rozen, F., Edery, I., Meerovitch, K., Dever, T. E., Merrick, W. C., and Sonenberg, N. (1990) Mol. Cell. Biol. 10, 1134-1144[Medline] [Order article via Infotrieve] |
39. | Merrick, W. C. (1979) Methods Enzymol. 60, 101-108[Medline] [Order article via Infotrieve] |
40. |
Grifo, J. A.,
Tahara, S. M.,
Leis, J. P.,
Morgan, M. A.,
Shatkin, A. J.,
and Merrick, W. C.
(1982)
J. Biol. Chem.
257,
5246-5252 |
41. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed , pp. 11.31-11.32, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
42. | Turner, D. H., and Sugimoto, N. (1988) Annu. Rev. Biophys. Biophys. Chem. 17, 167-192[CrossRef][Medline] [Order article via Infotrieve] |
43. | Sugimoto, N., Nakano, S., Katoh, M., Matsumura, A., Nakamuta, H., Ohmichi, T., Yoneyama, M., and Sasaki, M. (1995) Biochemistry 34, 11211-11216[Medline] [Order article via Infotrieve] |
44. | SantaLucia, J., Allawi, H. T., and Seneviratne, P. A. (1996) Biochemistry 35, 3555-3562[CrossRef][Medline] [Order article via Infotrieve] |
45. | Crooke, S. T. (1995) Therapeutic Applications of Oligonucleotides, p. 53-62, 123-130, R. G. Landes Co., Austin, TX |
46. | Altmann, K. H., Fabbro, D., Dean, N. M., Geiger, T., Monia, B. P., Muller, M., and Nicklin, P. (1996) Biochem. Soc. Trans. 24, 630-637[Medline] [Order article via Infotrieve] |
47. | Altmann, K. H., Nicholas, N. D., Fabbro, D., Freier, S. M., Geiger, T., Haner, R., Husker, D., Martin, P., Monia, B. P., Muller, M., Natt, F., Nicklin, P., Phillips, J., Pieles, U., Sasmor, H., and Moser, H. E. (1996) Chimia 50, 168-176 |
48. |
Freier, S. M.,
and Altmann, K. H.
(1997)
Nucleic Acids Res.
25,
4429-4443 |
49. |
Abramson, R. D.,
Dever, T. E.,
Lawson, T. G.,
Ray, B. K.,
Thach, R. E.,
and Merrick, W. C.
(1987)
J. Biol. Chem.
262,
3826-3832 |
50. | Goss, D. J., Woodley, C. L., and Wahba, A. J. (1987) Biochemistry 26, 1551-1556[Medline] [Order article via Infotrieve] |
51. |
Flores-Rozas, H.,
and Hurwitz, J.
(1993)
J. Biol. Chem.
268,
21372-21383 |
52. | Gross, C. H., and Shuman, S. (1996) J. Virol. 70, 2615-2619[Abstract] |
53. | Itoh, T., and Tomizawa, J. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2450-2454[Abstract] |
54. | Teplova, M., Minasov, G., Tereshko, V., Inamati, G. B., Cook, P. D., Manoharan, M., and Egli, M. (1999) Nat. Struct. Biol. 6, 535-539[CrossRef][Medline] [Order article via Infotrieve] |
55. | Hesson, T., Mannarino, A., and Cable, M. (2000) Biochemistry 39, 2619-2625[CrossRef][Medline] [Order article via Infotrieve] |
56. | Bianco, P. R., and Kowalczykowski, S. C. (2000) Nature 405, 368-372[CrossRef][Medline] [Order article via Infotrieve] |
57. | Salazar, M., Fedoroff, O. Y., Miller, J. M., Ribeiro, N. S., and Reid, B. R. (1993) Biochemistry 32, 4207-4215[Medline] [Order article via Infotrieve] |
58. |
Abramson, R. D.,
Dever, T. E.,
and Merrick, W. C.
(1988)
J. Biol. Chem.
263,
6016-6019 |