The poliovirus RNA-dependent RNA
polymerase binds cooperatively to single-stranded RNA. We have
determined the minimal RNA-binding site size of the poliovirus
polymerase using binding titration with oligonucleotides of increasing
length. A dramatic increase in affinity was observed when the length of
the oligo(U) increased from 8 to 10 nucleotides (nt), arguing that the
minimal size of RNA for polymerase binding is 10 nt. Another increase
in affinity seen as the oligo(U) reached 24 nt suggests that a
24-nucleotide RNA can be occupied by two polymerase molecules. Direct
binding of wild-type polymerase to oligo(U)12 and
oligo(U)24 RNAs showed differences in affinity and
cooperativity consistent with this model. The increase in binding
affinity seen for oligo(U)10 suggests either that the
RNA-binding determinants are widely spaced on the polymerase structure
or that a substantial conformational change in the polymerase occurs
upon the filling of its RNA-binding site.
 |
INTRODUCTION |
The poliovirus RNA-dependent RNA polymerase is
responsible for phosphodiester bond formation during templated RNA
polymerization. A 52-kDa protein, the poliovirus polymerase is encoded
by the 3'-most coding sequences of the poliovirus genome and is
generated by cleavage from the viral polyprotein by virally encoded
proteases. Studies of the mechanism of the poliovirus polymerase have
been greatly facilitated by its cloning (1) and expression both in
Escherichia coli (2-5) and in baculovirus-infected insect cells (6). Polymerase purified from these sources was identical in
elongation rate, template specificity, and Km for nucleotides to polymerase purified from infected cells (6).
The poliovirus polymerase displays a low affinity for RNA, which has
led to reports that highly purified polymerase is not an RNA-binding
protein (7, 8). However, recent in vitro studies using
poliovirus polymerase purified from E. coli demonstrated that at high concentration, the polymerase is not only capable of
binding RNA, but exhibits efficient template utilization (9). Surprisingly, interactions between poliovirus polymerase molecules themselves critically affect elongation activity in vitro.
Both RNA elongation and RNA binding were shown to be cooperative with respect to polymerase concentration (9). Data in support of polymerase-polymerase interactions have also come from observed interactions in the yeast two-hybrid system (10), from glutaraldehyde cross-linking (9), and from the three-dimensional structure of the
polymerase (11). An unusual feature of the poliovirus polymerase x-ray
crystallographic structure is an extensive interface between polymerase
molecules, termed Interface I (11). More than 23 amino acids are
involved in salt bridges, hydrogen bonds, and hydrophobic interactions
across Interface I. The packing of polymerase molecules at this
interface is head-to-tail, such that long fibers of polymerase extend
through the crystal lattice. To characterize further the RNA-binding
properties of poliovirus polymerase, we used competition with
homopolymeric RNAs of various lengths to determine the minimal site
size for polymerase binding to RNA.
 |
EXPERIMENTAL PROCEDURES |
Polymerase and Gene 32 Protein--
Wild-type poliovirus Mahoney
type 1 RNA-dependent RNA polymerase 3D proteins were
expressed from plasmids in E. coli BL21(DE3)pLysS under the
control of a T7 promoter and purified as described elsewhere (11),
except that immediately after elution from the final chromatography column, the polymerase preparation was brought to 62% glycerol. The
polymerase stocks, in 12.5 mM Tris-HCl (pH 8.0), 0.01%
NaN3, 0.05 mM EDTA, 0.25%
-octyl
glucopyranoside, 2 mM dithiothreitol, 120 mM
NaCl, and 60% glycerol, were stored at
80 °C. Wild-type polymerase was expressed from the plasmid pT5T-3D constructed by Thale
Jarvis (Ribozyme Products, Inc., Boulder, Colorado) and contained the
3D coding region preceded by an initiator methionine.
Purified gene 32 protein, expressed in E. coli from a
recombinant plasmid (12), was a generous gift from Dr. Y. Shamoo (Yale University). The gene 32 protein stock (89.4 µM) was
supplied in 10 mM Tris-HCl (pH 8.0), 50 mM
NaCl, 0.1 mM EDTA, 0.1 mM
-mercaptoethanol, and 10% glycerol and was stored at
80 °C.
Nucleic Acids--
Full-length positive-strand poliovirus RNA
was transcribed from T7pGEM-polio (13) digested with EcoRI.
The 116-nt 1 RNA was
transcribed from pG4Z-517/670 digested with BsaBI and encodes, from 5' to 3' of the T7-transcribed RNA, nt 1-29 derived from
the vector followed by nt 517-604 of the poliovirus Mahoney type 1 genome. The plasmid pG4Z-517/670 was constructed by inserting the
BstUI-BamHI fragment of T7pGEM-polio into pGEM4Z
(Promega, Madison, WI) digested with
HincII-BamHI. Transcription reactions were
carried out as described previously (9), except that UTP was included
at 0.5 mM.
Homopolymers were purchased from Pharmacia (Uppsala, Sweden) and
suspended in 10 mM Tris-HCl (pH 7.5) and 1 mM
EDTA. Uridylyl oligonucleotides 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 nt in length, as well as the 46-nt deoxyoligonucleotide
(ATAGTTCTGATCCACTCCGGGCCCTACAGGTCATACTGTAATTGCC), were synthesized by
Macromolecular Resources (Fort Collins, CO). The sequence of the 46-nt
DNA was derived from the yeast Saccharomyces cerevisiae
double-stranded RNA virus L-A, nt 4506-4551 (14), and thus should
represent a heteropolymeric DNA of irrelevant sequence. Thymidyl
deoxyoligonucleotides 3, 4, 5, 6, 7, 8, 9, 10, and 11 nt in length were
synthesized by the Protein and Nucleic Acid facility at Stanford
University.
For the direct binding of oligo(U)12 and
oligo(U)24 RNAs and the 46-nt DNA, the nucleic acids were
end-labeled with 50 µCi of [
-32P]ATP (3000 Ci/mmol;
NEN Life Science Products) with 1 unit/µl T4 polynucleotide kinase
(New England Biolabs Inc.) in 70 mM Tris-HCl (pH 7.6), 10 mM MgCl2, and 5 mM dithiothreitol.
Unincorporated [
-32P]ATP was removed by centrifugation
through a Micro-spin G-25 column (Pharmacia) once for the DNA and twice
for the RNA. Concentration was determined by UV absorption at 260 nm.
Direct Binding and Competition Reactions--
Polymerase at
concentrations ranging from 0.20 to 7.0 µM was incubated
with the appropriate 32P-labeled nucleic acid in 50-µl
reactions with final concentrations of 25 mM MES-NaOH (pH
5.5), 60 mM NaCl, 5 mM MgCl2, 0.1 mM ZnSO4, 5 mM dithiothreitol, 0.25 mM ATP, 0.25 mM UTP, 0.25 mM GTP,
0.25 mM CTP, and 24% glycerol. Polymerase binding to RNA
and enzymatic activity were previously found to be optimal at pH 5.5 under these conditions (9). Reactions were incubated on ice for 30 min. Full-length poliovirus RNA and the 116-nt RNA were present at final
concentrations of 0.01 and 1 nM strands, corresponding to 75 and 116 nM nt, respectively. Oligo(U)12 and
oligo(U)24 were present at 12 and 6 nM strands,
respectively, corresponding to 144 nM nt.
For competition experiments, unlabeled competitor RNAs at various
concentrations were mixed with the radioactively labeled RNA in binding
reaction buffer as described above. Polymerase at either 1.5 µM for the full-length poliovirus RNA or 2.0 µM for the 116-nt RNA was added to a final volume of 50 µl, and the competition reactions were allowed to equilibrate on ice
for 30 min.
Gene 32 protein in concentrations ranging from 0.12 to 3.0 µM was incubated with the appropriate
32P-labeled nucleic acid in a 25-µl solution containing 5 mM MES-NaOH (pH 6.5), 2 mM Tris-HCl (pH 8.0),
10 mM NaCl, and 0.35 mM MgCl2 and
incubated at room temperature for 30 min. The final pH of this solution
was 7.5. The 116-nt RNA and the 46-nt DNA were present at final
concentrations of 1 and 2 nM strands, corresponding to 116 and 92 nM nt, respectively.
For competition experiments, unlabeled competitor oligo(dT) molecules
at various concentrations were mixed with the radioactively labeled RNA
or DNA in binding reaction buffer as described above. Gene 32 protein
at either 0.60 µM for the 116-nt RNA or 0.22 µM for the 46-nt DNA was added to 25 µl, and the
competition reactions were allowed to equilibrate for 30 min at room
temperature.
Filter Binding Assay--
Complexes present in aliquots of the
polymerase-nucleic acid binding reactions were separated by a modified
nitrocellulose binding assay described previously (9). For the
polymerase binding experiments, the filters were washed with 500 µl/well of a solution containing 50 mM Hepes-NaOH (pH
7.5) and 5 mM MgCl2. When measuring the direct
binding of polymerase to 32P-labeled oligo(U)12
and the 116-nt RNA in Fig. 5, the filters were washed with 2 ml/well of
buffer containing 25 mM MES-NaOH (pH 5.5), 5 mM
MgCl2, 0.1 mM ZnSO4, and 5 mM dithiothreitol.
Separation of gene 32 protein-nucleic acid complexes was performed
using a similar binding assay with only the nitrocellulose filter, to
capture the gene 32 protein-nucleic acid complexes, and the positively
charged nylon filter, to capture the unbound nucleic acids. Complexes
bound to the membranes were washed with 1 ml/well of a solution
containing 50 mM Hepes-NaOH (pH 7.5) and 5 mM
MgCl2. Detection and quantitation of the radiolabeled
nucleic acids bound to the filters were performed using a Storm
PhosphorImaging System with ImageQuant software (Molecular Dynamics,
Inc., Sunnyvale, CA).
Data Analysis--
All graphing and curve fitting were performed
using GraphPAD Prism software (GraphPAD Software for Science, San
Diego, CA). Data points for direct binding of nucleic acids to proteins
were collected in duplicate or triplicate at each protein
concentration. The mean values are given in the figures, with error
bars representing S.D.
Direct binding curves that displayed cooperative binding behavior with
respect to polymerase concentration were fit to Equation 1, based on
the Hill equation.
|
(Eq. 1)
|
The exponential term (n) is the Hill coefficient,
which can be used as a rough estimate of the extent of cooperativity
(15). However, since gene 32 protein binding saturated with <100% of the input nucleic acid bound, the amount of nucleic acid bound at
saturation was estimated, and Equation 1 was modified to reflect this
(Equation 2).
|
(Eq. 2)
|
Direct binding curves that did not display cooperative binding
behavior with respect to polymerase concentration were fit to a simple
binding curve derived from the law of mass action, where Equation 3
follows,
|
(Eq. 3)
|
and Bmax is the maximum percent RNA
bound, [protein] is the micromolar concentration of the protein, and
Kd is the dissociation constant. To determine
whether direct binding curves displayed simple or cooperative binding,
all curves were fit to both types of equations, and the correlation
coefficients (R2 values) were compared. The
binding curves that gave rise to the highest R2
values were chosen for presentation.
Competition data were fit to Equation 4, which describes competition
between bound ligand and competitor for binding to the same site.
|
(Eq. 4)
|
Values of IC50, the concentration of competitor at
which binding of labeled heteropolymeric RNA was reduced by 50%, were determined by nonlinear regression analysis. Error bars shown for the
values of IC50 as a function of oligonucleotide competitor length show the 90% confidence interval for each IC50
value, calculated from the standard error of the individual competition
curves.
 |
RESULTS |
Experimental Rationale--
These experiments were performed to
determine the minimal size of RNA that could be bound by the poliovirus
RNA-dependent RNA polymerase. We tested the ability of
small RNA oligonucleotides of different lengths to compete for binding
of poliovirus polymerase to a 116-nt labeled RNA to which multiple
polymerase molecules bind cooperatively (9). The effectiveness of
competition is measured as the IC50, the concentration of
RNA oligonucleotide (in nucleotides) at which binding to the 116-nt RNA
is inhibited by 50%. The IC50 values are linearly related
to the Kd and therefore reflect the binding energy
of the complex between the competing RNA oligonucleotide and
polymerase.
Two outcomes are possible, depending on the nature of the
polymerase-RNA interaction. If a protein binds to RNA noncooperatively or binds to RNA cooperatively but without an appreciable change in
binding energy when its entire RNA-binding site is filled, the outcome
shown in Fig. 1A will be
observed. In this case, the affinity of RNA oligonucleotides smaller
than a single binding site and those that can accommodate only one
protein molecule will be approximately a function of the number of
nucleotides, with each nucleotide contributing equal binding energy.
Then, when the RNA oligonucleotide is long enough to accommodate two proteins, an increase in binding affinity that reflects the energy of
the protein-protein interaction may or may not be observed.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Predicted outcomes of oligonucleotide
competition experiments to determine the minimal binding site size of a
single-stranded nucleic acid-binding protein. A, effect of
oligonucleotide length on binding energy/nucleotide of protein-RNA
complex if each nucleotide contributes equal binding energy and no
significant contribution to binding energy is derived from the filling
of the RNA-binding site. B, effect of oligonucleotide length
on binding energy/nucleotide of protein-RNA complex if changes in
overall affinity of the protein-RNA complex occur upon the filling of the RNA-binding site. 0, no bound polymerase; 1,
one bound polymerase molecule; 2, two bound polymerase
molecules. Circles, bound polymerase; irregularly
shaped circle, unbound polymerase; ------, RNA.
|
|
A second possible outcome would be observed if the energy/nucleotide of
the protein-RNA interaction were to increase when the RNA-binding site
was filled (Fig. 1B). One possible reason for this effect
would be a conformational change in the protein upon binding to a
complete site. Large apparent conformational changes upon DNA binding
have been reported for human immunodeficiency virus type 1 reverse
transcriptase and RecA (16-18). Another example of large increases in
binding affinity with only small increases in oligonucleotide length
was seen in the binding of E. coli cyclic AMP-binding
protein to small DNA molecules (19). In this case, DNA-binding
determinants are located on opposite sides of single cyclic AMP-binding
proteins. Once a DNA molecule is large enough to contact both sites
simultaneously, its binding affinity for cyclic AMP-binding protein
increases dramatically (19). If either of these effects were to
contribute additional energy to the binding of small homopolymeric RNAs
long enough to fill the binding site of poliovirus polymerase,
comparing the effectiveness of competition of different length
oligonucleotides would be a useful technique to measure the binding
site size. This is the approach taken in the experiments described
below.
Competition of Polymerase Binding to Poliovirus RNA by Long
Homopolymeric RNAs--
Poliovirus polymerase has been shown to bind
to heteropolymeric RNA with little sequence specificity and relatively
low affinity, but high cooperativity (Ref. 9 and data not shown). Fig.
2A shows the highly
cooperative binding of purified polymerase to full-length transcripts
of poliovirus RNA. The lack of sequence specificity of polymerase
binding makes it reasonable to use homopolymeric RNAs to compete with
heteropolymeric RNAs for polymerase binding. Fig. 2B shows
that poly(C) and poly(U) proved to be good competitors, whereas neither
poly(G) nor poly(A) competed effectively with heteropolymeric RNA for
polymerase binding under these conditions, chosen to optimize the
cooperativity of polymerase binding (9). The poor competition observed
with polypurines could be due to alternative structures formed by
polypurines. Competition experiments with oligonucleotides of different
lengths, described below, employed uridylyl oligonucleotides of varying
size.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Cooperative binding of polymerase to
poliovirus RNA and competition by long homopolymeric RNAs.
A, percentage of total poliovirus RNA bound as a function of
increasing polymerase concentration. The theoretical curve (------) for
cooperative binding (see "Experimental Procedures," Equation 1)
gives values of n = 5.7, K = 9.6 µM5.7, and R2 = 0.98. B, competition of polymerase binding to poliovirus RNA by
homopolymeric RNAs. Only one point for poly(A) and one point for
poly(G) are shown for clarity. Theoretical curves assume competition between bound ligand and competitor for binding to the same site (see
"Experimental Procedures," Equation 2). , poly(U),
R2 = 0.98; , poly(C),
R2 = 0.95; , poly(G); , poly(A).
|
|
Competition of Polymerase Binding to 116-nt Heteropolymeric RNA by
Oligo(U) of Varying Lengths--
To test the effect of oligonucleotide
length on its affinity for poliovirus polymerase, oligo(U) molecules of
varying lengths thought to span a range of sizes smaller than one
polymerase-binding site, sufficient to bind one polymerase molecule,
and perhaps long enough to bind two or multiple polymerase molecules
were synthesized. The recently determined structure of poliovirus
polymerase has revealed that the direct distance between corresponding
residues in the active sites of adjacent polymerase molecules in the
crystal is ~46 Å (11). An observed correlation between cooperative
RNA binding by polymerase and polymerase activity has led us to
hypothesize that the template for poliovirus polymerase is
single-stranded RNA complexed with cooperatively bound polymerase
molecules (9). If polymerase molecules can bind head-to-tail in a fiber
along regions of an RNA molecule, the bound single-stranded RNA is
expected to traverse the length of each polymerase molecule. Depending on the conformation of the bound RNA, the length of RNA bound to a site
that is at least 46 Å in length could range from 16 or more
nucleotides for an A-form conformation to as few as 8 nt for an
extended single-stranded conformation. These values are based on
observed internucleotide distances of ~6.0 Å/nt for the highly
extended single-stranded RNAs bound to U1A spliceosomal protein (20),
of ~5.0 Å/nt for the single-stranded DNA bound to bacteriophage T4
gene 32 protein (21), and of 2.8 Å/base pairs for A-form RNA (22).
Fig. 3A shows the cooperative
binding of poliovirus polymerase to a 116-nt RNA derived from the
poliovirus genome. This RNA, considerably smaller than the full-length
viral RNA used in Fig. 2, was used to avoid masking the effect of
competitor oligonucleotides by the high degree of cooperativity seen
with very long RNAs. Half-maximal binding of polymerase to the 116-nt
RNA occurred at ~3 µM polymerase 3D. Competition
experiments were then performed using a lower concentration of
polymerase (2 µM) to maximize sensitivity of the
competition assay. Oligo(U) molecules 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 nt in length were tested for their affinities for poliovirus
polymerase in competition experiments against the 116-nt RNA. Fig.
3B shows individual competition curves of
oligo(U)6-24. The individual data points were fit to an
equation that describes competition for binding to one site. The
cross-hairs in each graph denote the IC50 in nanomolar
nucleotides. A lower IC50 value indicates greater affinity
for the polymerase.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Cooperative binding of polymerase to 116-nt
heteropolymeric RNA and competition by oligo(U) of different lengths.
A, percentage of total 116-nt RNA bound as a function of
increasing polymerase concentration. Equation 1 (------) gives values
of n = 2.2, K = 10.8 µM2.2, and R2 = 0.99. B, competition of polymerase binding to the 116-nt RNA by
oligo(U) of different lengths. For each oligo(U),
cross-hairs depict IC50 (in nanomolar
nucleotides).
|
|
For lengths smaller than 8 nt, the RNAs did not compete effectively
(Fig. 3B). In fact, conditions under which competitor oligo(U)4 (data not shown) and oligo(U)6
displayed any competition, and therefore any measurable binding to
polymerase, were not achieved. When the length of the oligo(U) reached
10 nt, however, substantial competition for binding to the 116-nt
labeled RNA was seen. Little further increase in the ability to compete
was observed until the longest RNA oligonucleotide,
oligo(U)24, was tested.
RNA-binding Site Size--
When the IC50 values
determined in Fig. 3B are plotted as a function of
oligonucleotide length, it is clear that appreciable binding to
polymerase was not observed until the RNA reached a length of 10 nt
(Fig. 4). The drop in IC50 at
10 nt is substantial: the addition of 2 nt to an RNA oligonucleotide of
8 nt decreased the IC50 by ~20-fold, and the addition of
2 nt to an RNA oligonucleotide of 10 nt decreased the IC50
by only 2-fold. It is not likely that the change in affinity for
polymerase at 10 nt represents the length of RNA to which two
polymerase molecules can be cooperatively bound, both because the site
size of cooperative polymerase binding is likely to be at least 8 nt to
span between polymerase active sites and because no appreciable binding
affinity was observed for RNA oligonucleotides smaller than 10 nt in
length. We consider it likely that the RNA-binding site is ~10 nt and
that a binding site that is fully occupied has much greater affinity
for its RNA substrate than a binding site that is only partially filled by its RNA substrate.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
IC50 values decrease as a
function of oligonucleotide length. IC50 values from
the experiments shown in Fig. 3B are plotted as a function
of oligo(U) length. Error bars represent the 90% confidence
interval determined from each individual data set.
|
|
The IC50 values decrease somewhat linearly with the length
of the oligo(U) from oligo(U)12 to oligo(U)22.
Modest increases in affinity with increasing oligonucleotide lengths,
even if only one polymerase can be bound to each oligonucleotide, are
expected due to "lattice effects": in a longer polymer, there are
more ways to occupy a site of defined length than in a shorter one (24). At 24 nt, a larger apparent increase in oligo(U) affinity was
seen that is likely to correspond to the addition of a second polymerase molecule (Fig. 4).
Direct Binding of Polymerase to Oligo(U)12 and
Oligo(U)24--
If the minimal binding site for a single
polymerase is 10-12 nt in length, then polymerase binding to oligo(U)
molecules that contain only a single binding site may reveal the
intrinsic affinity of polymerase for RNA, separated from the
protein-protein interactions that are likely to dominate the energetics
of polymerase binding to longer RNAs. Direct binding of polymerase to
oligo(U)24, predicted to contain two binding sites for
polymerase, would then reflect the intrinsic affinity of two polymerase
molecules for RNA as well as the protein-protein interactions between
the polymerases. Fig. 5 compares the
direct binding of wild-type polymerase to oligo(U)12 and
oligo(U)24. The affinity of polymerase for
oligo(U)12 was much less than half of that seen for
oligo(U)24. Furthermore, the apparent cooperativity of
binding to oligo(U)12 was less than binding to
oligo(U)24 (Fig. 5). While these observations are
consistent with the hypothesis that one polymerase molecule binds to
oligo(U)12 and two polymerase molecules bind cooperatively
to oligo(U)24, it is interesting that the binding of
polymerase to oligo(U)12 still displayed some
cooperativity. It may be that interactions between RNA-bound polymerase
and free polymerase can increase the strength of RNA binding.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Direct binding of wild-type polymerase to
oligo(U)12 and oligo(U)24. Shown is the
percentage of total oligo(U)12 and oligo(U)24
bound as a function of polymerase concentration. The data were fit to
Equation 1 with a maximum RNA bound of 77% and give values of
n = 1.4, K = 24 µMn, and R2 = 0.98 for
oligo(U)12 and n = 2.8, K = 2.3 µMn, and R2 = 0.98 for
oligo(U)24. , oligo(U)24; ,
oligo(U)12.
|
|
Competition of Gene 32 Protein Binding to RNA and DNA
Polynucleotides by DNA Oligonucleotides of Different Lengths--
To
test the method of titration with oligonucleotides of increasing length
on a well known cooperative single-stranded binding protein, we tested
the effect of oligonucleotide competition on the binding of
bacteriophage T4 gene 32 protein to both the 116-nt RNA and a 46-nt
heteropolymeric single-stranded DNA. Site sizes of binding for gene 32 protein monomers from 5 to 10 nt have been reported (21, 24-26), with
a consensus at 6 nt in recent literature (21). The extent of
cooperativity can be varied by changing the binding conditions: the use
of RNA-binding substrates, for example, is known to disfavor
cooperative binding under many conditions (26, 27).
A simple binding curve of gene 32 protein to the 116-nt RNA at
relatively low salt concentrations is shown in Fig.
6A. The noncooperative nature
of this binding (n = 1.0) is readily seen. The effect
of competing the RNA-binding signal observed at 0.6 µM
gene 32 protein with varying lengths of oligo(dT) (Fig. 6B) and the derived IC50 values as a function of
deoxyoligonucleotide length (Fig. 6C) were determined. The
IC50 values remained relatively constant over the range of
deoxynucleotide lengths tested.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 6.
Noncooperative binding of gene 32 protein to
116-nt heteropolymeric RNA and competition by oligo(dT) of different
lengths. A, percentage of total 116-nt RNA bound as a
function of increasing gene 32 protein concentration. The theoretical
curve describes simple binding where Bmax = 29%
RNA bound, K = 1.3 µM, and
R2 = 0.97. B, competition of gene 32 protein binding to the 116-nt RNA by oligo(dT) of different lengths.
For each competition curve, cross-hairs depict
IC50 (in nanomolar nucleotides). C,
IC50 values from B plotted as a function of
oligo(dT) length. Error bars represent the 90% confidence
interval determined from each individual data set.
|
|
To test whether DNA oligonucleotides could be bound cooperatively under
these conditions, the binding of gene 32 protein to a 46-nt
single-stranded DNA heteropolymer was performed (Fig. 7A). A simple binding curve
proved to be highly cooperative with respect to gene 32 protein
concentration (n = 2.7). When the DNA-binding signal
observed at 0.22 µM gene 32 protein was competed using oligo(dT) molecules of increasing length (Fig. 7B), very
little effect of deoxyoligonucleotide length on the IC50
values was again observed (Fig. 7C). The IC50
values plotted against deoxyoligonucleotide length resulted in the
graph shown in Fig. 7C. A small drop in IC50 was
reproducibly observed between oligo(dT)8 and
oligo(dT)9.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 7.
Cooperative binding of gene 32 protein to
46-nt heteropolymeric DNA and competition by oligo(dT) of varying
lengths. A, percentage of total 46-nt DNA bound as a
function of increasing gene 32 protein concentration. The cooperative
binding curve (see "Experimental Procedures") had a maximum of 29%
DNA bound and gives values of n = 2.7, K = 0.025 µM2.7, and
R2 = 0.93. B, competition of gene 32 protein binding to the 46-nt DNA by oligo(dT) of different lengths. For
each oligo(dT), cross-hairs depict IC50 (in
nanomolar nucleotides). C, IC50 values from the experiments in B plotted as a function of oligo(U) length.
Error bars represent the 90% confidence interval determined
from each individual data set.
|
|
 |
DISCUSSION |
Significance of Cooperative Binding of Poliovirus Polymerase to
Single-stranded RNA to the Poliovirus Replicative Cycle--
The
expression strategy of all picornaviruses, including poliovirus,
dictates that approximately equimolar amounts of viral enzymes (such as
proteases and the RNA-dependent RNA polymerase 3D) and
capsid proteins are synthesized. Sixty molecules of each capsid
proteins are required to encapsidate each RNA genome. Thus, given the
proportionately large quantities of polymerase available, it is
possible that the 3D polypeptide also functions as a single-stranded binding protein.
Thus far, the poliovirus polymerase has not been reported to bind
specifically to any RNA sequence. However, any specificity could easily
have been masked by the high degree of cooperativity. Now it can be
appreciated that fairly small RNAs must be screened for those that
contain specific polymerase-binding sites that might nucleate RNA
binding. The single-stranded RNA-binding activity could play numerous
roles in the infectious cycle, as suggested by the functions served by
other single-stranded RNA-binding proteins. For example, T4 gene 32 protein has been shown to decrease the error rate of T4 DNA polymerase
(21, 28), and RecA is absolutely required for replication and
recombination of the E. coli genome (29). Gene 32 protein
down-regulates the translation of its own mRNA by binding to an RNA
pseudoknot that includes the initiating AUG codon (30). A role for a
single-stranded nucleic acid-binding protein in the packaging of
single-stranded nucleic acid into virions has been established for gene
V protein of M13 phage, which binds to newly synthesized
single-stranded phage DNA molecules, preventing their further
replication and transferring them, by an as yet unknown mechanism, to
the empty phage capsid (reviewed in Ref. 31).
The homo-oligomerization of polymerase molecules has no precedent in
the DNA-dependent polymerases. Among the reverse
transcriptases, human immunodeficiency virus type 1 reverse
transcriptase forms a heterodimer between one full-length and
one proteolytically processed subunit (32, 33); Moloney murine leukemia
virus reverse transcriptase has been reported to show some cooperative binding to DNA templates and is likely to act as a dimer (34). Whether
the cooperative binding of poliovirus RNA-dependent RNA polymerase to RNA is shared with the polymerases of other
positive-stranded RNA viruses, such as the closely related rhinoviruses
and the more distantly related hepatitis C virus, remains to be
tested.
Stoichiometry of Polymerase Binding to RNA--
Does the low
efficiency of RNA binding by purified poliovirus polymerase reflect a
low affinity for RNA templates, or does it reflect the activity of only
a subset of the purified polymerase molecules? For each of the
experiments presented here, micromolar concentrations of polymerase
were required to saturate complex formation with labeled RNAs present
at nanomolar concentrations. For example, in Fig. 3A, ~3
µM polymerase was required to retain half of the 116-nt
labeled RNA, present at 1 nM strands, in protein-RNA complexes on nitrocellulose filters. The likelihood that multiple polymerases bind to each RNA is obviously not sufficient to explain the
necessity for a 3000-fold molar excess of polymerase for half-maximal RNA binding. Titration of complex formation between polymerase, present
at 2 µM, with increasing amounts of the 116-nt RNA has shown that complex formation saturates at ~0.2 µM RNA,
at a polymerase/RNA ratio of 10:1 (data not shown). Therefore, there is
not likely to be a large population of polymerase that is inactive in
RNA binding. Despite the low affinity of poliovirus polymerase for its
RNA substrates, it is possible that, within infected cells, other viral
or host proteins in the replication complex serve to increase the
affinity and specificity of poliovirus polymerase for RNA (8, 10,
35-37). For poliovirus polymerase, understanding the nature of the
contacts made between the polymerase and RNA in isolation will give us
a starting point to understand the function of accessory proteins in
the RNA replication complex.
The Binding Site for a Single Polymerase Molecule Is 10-12 nt in
Length--
Due to the low affinity of poliovirus polymerase for RNA,
and especially for small oligonucleotides (Fig. 5), we used competition experiments to test the affinities of RNA oligonucleotides of different
lengths (Figs. 3 and 4). RNA oligonucleotides 10 nt and longer show a
large increase in affinity for poliovirus polymerase, as judged by
their ability to compete with longer heteropolymeric RNAs for
polymerase binding (Fig. 4). This could result from one of two possible
effects of oligo(U) length. We favor the interpretation that, at 10 nt,
the RNA has reached a length that can completely or almost completely
occupy the RNA-binding site in one polymerase molecule. There are
several arguments that favor this interpretation. First, 10 nt could
readily span the 46-Å distance between polymerase active sites, given
two polymerase molecules juxtaposed at the substantial interface
(Interface I) shown in the three-dimensional structure (11). Depending
on the path of the RNA between polymerase molecules, the actual
distance traversed is likely to be greater than the direct distance of
46 Å. The internucleotide distance of the bound RNA traversing the
polymerase molecule would then be, on average, at least 4.6 Å/nt,
comparable to similar distances observed in complexes of RNA- and
DNA-binding proteins with single-stranded nucleic acids (38, 39).
Second, another substantial drop in IC50 was not observed
until another 12-14 nt were added to the length of the oligo(U)
competitor (Fig. 4). It is likely that competition with
oligonucleotides can result in an underestimation of the actual site
size because bound proteins can occlude more nucleotides than they
interact with physically (40).
Direct binding of polymerase to oligo(U)12 and
oligo(U)24 (Fig. 5) supported the hypothesis that a single
polymerase molecule could bind to oligo(U) molecules 10-12 nt in
length, and 24 nt was sufficient to span two polymerase molecules.
Polymerase binding to oligo(U)24 was highly cooperative and
showed an apparent affinity much greater than twice that observed for
binding to oligo(U)12 (Fig. 5).
Another interpretation of the decrease in IC50 observed
with oligo(U)12 is that, when the oligo(U) reaches 10-12
nt in length, two polymerase molecules can bind. If this were the case,
another drop in IC50 at 16-18 nt might be expected due to
binding of a third polymerase; this was not observed (Fig. 4). To span
46 Å from active site to active site, even at the very extended
single-stranded configuration of 6 Å/nt observed in the complex of
single-stranded RNA with the U1 small nuclear ribonucleoprotein (Code
1URN, Protein Data Bank, Brookhaven National Laboratory (20)), would
require at least 8 nt.
Conformational Change in Polymerase upon RNA Binding--
The
20-fold decrease in IC50 from oligo(U)8 to
oligo(U)10 is likely to reflect either a large
conformational change upon binding those RNA molecules that can
completely occupy the RNA-binding site of the polymerase or the
existence of widely spaced binding determinants in the RNA-binding
site. A large conformational change upon substrate binding has been
documented for human immunodeficiency virus type 1 reverse
transcriptase, for example. Compared with the crystal structure of the
free enzyme (32), the thumb domain of human immunodeficiency virus type
1 reverse transcriptase is pivoted away from the fingers domain by 30 Å in the structure of a co-crystal with a template-primer DNA (16).
Other possible conformational changes upon RNA binding might involve
increased affinity for other polymerase molecules. Widely spaced
binding determinants are thought to be responsible for the extreme
sensitivity of cyclic AMP-binding protein to DNA length (19).
Interestingly, little inflection in the IC50 values was
observed when T4 gene 32 protein binding to either RNA or DNA was competed with oligo(dT) of varying lengths that spanned the 6-nt site
size. An artifactual explanation of this observation might be that,
when gene 32 protein is in "oligonucleotide-binding mode," it binds
only to the ends of small oligonucleotides and is insensitive to their
length or base composition (26). If this were the case in Figs. 6 and
7, the IC50 (in nanomolar nucleotides) should increase with
increasing oligonucleotide length, which it does not. Rather, it seems
likely that the nucleic acid-binding site of gene 32 protein can bind
regions of single-stranded nucleic acid shorter than its complete
binding site with as high an affinity/nucleotide as to complete sites.
The three-dimensional structure of gene 32 protein in the absence of
the protein-protein interaction domains shows 4 nt of DNA resolved in
the binding site of the "core" protein. The other 2 nt in the 6-nt
DNA site are thought to be contacted less closely by gene 32 protein
and are disordered in the crystal (21). The ability to fill a binding
site partially might be useful in the function of gene 32 protein in
stabilizing the unfolded state of partially single-stranded DNA
molecules during replication, recombination, and repair. The structure
of gene 32 protein in the absence of complexed DNA has not been
determined, so any conformational changes that occur upon nucleic acid
binding have not been characterized structurally. However, gene 32 protein contains an N-terminal peptide that is thought to occupy the
nucleic acid-binding site in the free protein, but to move out of the
binding cleft when it is occupied by nucleic acid (41). Then, this
N-terminal peptide, which is required for the cooperativity of
single-stranded nucleic acid binding, is free to interact with other
gene 32 protein molecules. In this context, the inflection in
IC50 at 9 nt (Fig. 7C) is interesting: it is
perhaps not until another gene 32 protein monomer can be contacted that
any increase in energy as a function of oligonucleotide length can be
realized. In any case, the gene 32 protein studies reveal that the
method of oligonucleotide titration is a useful method to determine
site size of protein binding only for certain proteins, probably those
that undergo large conformational changes upon full occupation of their
nucleic acid-binding sites or that contain widely spaced binding
determinants.
We thank Yousif Shamoo for supplying the gene
32 protein used in this study; Kay Sanders for assistance with protein
purification; Peter Sarnow, Steve Schultz, and John Lyle for critical
reading of the manuscript; and John Lyle for assistance with the
figures. We are especially grateful to Steve Schultz and Jeff Hansen
for extensive discussions of the three-dimensional structure of
poliovirus polymerase.