From the Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131
Received for publication, March 17, 2001
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ABSTRACT |
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PP7 is a single-strand RNA bacteriophage
of Pseudomonas aeroginosa and a distant
relative to coliphages like MS2 and Q The coat proteins of several single-strand RNA bacteriophages are
known translational repressors. They shut off viral replicase synthesis
by binding an RNA hairpin that contains the replicase ribosome binding
site. Recent x-ray structure determination of RNA phages shows that
homologies evident from comparisons of coat protein amino acid
sequences are reflected in their tertiary structures (1-7). The coat
protein dimer, which is both the repressor and the basic building block
of the virus particle, consists of two intertwined monomers that
together form a large Plasmid Constructions--
The PP7 coat sequence cloned on a
plasmid was kindly provided to us by Gordon Garde. We amplified the
coat sequence using polymerase chain reaction and a 5'-primer
(5'-GGGTCTAGACGTTACAGCGACTACTGAAACGTAAG-3') that introduced a
XbaI site about 40 nucleotides upstream of the coat
initiation codon, and a 3' primer
(5'-GGGGGATCCATACACACGGGTACACCGCAGGGCC-3') that created a
BamHI site a few nucleotides downstream of the stop
codon. This and subsequent amplifications were conducted using
Pfu DNA polymerase. After digestion with XbaI and
BamHI, the fragment was cloned between the corresponding
restriction sites within the polylinker of pUC119 (11), placing the
coat sequence under control of the lac promoter. We call
this plasmid pP7CT. Because of relatively low expression of the PP7
coat protein from pP7CT, we constructed another plasmid we called
pETP7CTNc. This was accomplished by
PCR1 amplification of the
coat sequence using the same BamHI site-containing 3' primer
described above, and a new 5' primer
(5'-GACACCATGGCCAAAACCATCGTTCTTTCGGTCGG-3'), which introduced a
NcoI site at the initiator AUG of the coat gene. After
digestion with NcoI and BamHI, this fragment was
inserted into pET3d (12), where it became attached to the T7 promoter and gene 10 ribosome binding site. Plasmid pETP7CTNc produces large
amounts of PP7 coat and is a convenient source of the protein. However,
it is unsuitable for translational repressor assays in our two-plasmid
system. Therefore, we constructed pP7CTNcXb by transfer of the
XbaI-BamHI fragment from pETP7CTNc to pUC119. This construction retains the fusion of coat to the T7 gene 10 ribosome
binding site, but returns coat to lac promoter control in pUC119.
The plasmid we call pRZP7 is similar to the previously constructed pRZ5
(13). A synthetic EcoRI-BamHI fragment containing the putative PP7 translational operator replaces the corresponding MS2
operator sequence of pRZ5. This fuses the Escherichia coli lacZ gene to a synthetic version of sequences surrounding the PP7
replicase start codon, which we assumed contain the PP7 translational operator (14). This sequence is shown in Fig. 1. The object of this
construction was to place the synthesis of Protein Preparation and Reduction of Disulfide Bonds--
To
determine whether coat protein contains interchain disulfide bonds, PP7
capsids produced in E. coli from either pETP7Nc or pP7CTNcXb
were purified on Sepharose CL-4B by methods described previously (13),
and incubated at a concentration of 1 mg/ml in 50 mM Tris,
pH 8.5, with DTT at concentrations varying from 0 to 50 mM
at 0 °C. After 60 min, reactions were terminated by addition of
N-ethylmaleimide to a concentration of 200 mM.
Samples were then subjected to electrophoresis under non-reducing
conditions on 12.5% polyacrylamide gels containing SDS. Protein was
visualized by staining with Coomassie Brilliant Blue.
Coat protein was prepared for RNA binding studies by incubation in 50 mM Tris, pH 8.5, 7 M urea, 5 mM DTT
at 0 °C for 60 min. followed by dialysis against 1 mM
acetic acid (about pH 4). Renatured coat protein was stored at 4 °C,
where it appears to be stable for several weeks.
Measuring Translational Repression and RNA
Binding--
Nitrocellulose filter binding was performed using a
Schleicher & Schuell dot-blot apparatus and the method of Wong and
Lohman (15). RNAs were labeled with [
Translational repression was assessed by the ability of
coat proteins expressed from pCT119, pP7CTNcXb, and pQCT to inhibit the
synthesis of Mapping the RNA Binding Site--
Using site-directed
mutagenesis (17) of single-stranded pP7CTNcXb, we constructed libraries
of mutations directed to specific codons for amino acids potentially
involved in RNA binding. We used degenerate oligonucleotide primers
which converted target codons to NNG/T, thus resulting in the possible
introduction of all 20 amino acids and one stop codon. DNA from
mutagenesis reactions was introduced by transformation into strain
CSH41F-(pRZP7) and plated at a densities of about 500 colonies/plate on
solid LB medium containing
5-bromo-4-chloro-3-indolyl- Construction of Plasmids for Overexpression of Coat Protein and in
Vivo Assay of Its Translational Repressor Activity--
The
expectation that PP7 coat protein would repress the translation of
replicase was only an extrapolation from our understanding of the
behaviors of better characterized RNA coliphages like MS2 and Q
To test for translational repression in vivo, we constructed
a two-plasmid system for PP7. The first plasmid, analogous to the MS2
coat protein producer pCT119 (8, 13), expresses PP7 coat protein from
the lac promoter on a plasmid that confers ampicillin resistance. We PCR-amplified the coat sequence using primers that introduced a XbaI site about 40 nucleotides upstream of the
coat protein start codon and a BamHI site just downstream of
its stop codon. The resulting XbaI-BamHI fragment
was inserted into pUC119, resulting in the plasmid we call pP7CT.
A second plasmid, pRZP7, was constructed by the fusion of a synthetic
sequence containing the putative PP7 translational operator to a
deletion mutant of the
From the beginning, translational repression was evident from the
blueness (or, better, the relative lack of blueness) of colonies
containing the two plasmids on X-gal plates. However, compared with the
MS2 and Q
The plasmid pETP7CTNc provides a convenient source of PP7 coat protein,
which can be purified by methods described in this paper and elsewhere
(13), but it is unsuitable for the translational repression assays we
usually perform in strain CSH41F Disaggregation of Capsids and Refolding of PP7 Coat Protein for RNA
Binding Studies in Vitro--
We have previously prepared the coat
proteins of the various phages by purifying virus-like particles by
chromatography on Sepharose CL4B (13). These were then subjected to
denaturation in 50% acetic acid and dialyzed against 1 mM
acetic acid (about pH 4). These treatments generally result in
disaggregation of capsids and subsequent renaturation of coat protein
dimers without reassembly into capsids. This procedure is necessary
because the RNA binding site of the RNA phage coat proteins is
inaccessible on the interior surface of intact virus-like particles.
Unfortunately, when we applied these methods to PP7, they resulted in a
coat protein preparation that RNA-excess titrations indicated was no more than 0.15% active (results not shown). Thus, we sought an alternative disaggregation/renaturation protocol.
We noted the presence of two cysteine residues in the FG loop of PP7
coat protein and wondered whether they were involved in the formation
of interchain disulfide bonds in a manner similar to Q
We tried several disaggregation protocols, including one in
which we employed the minimum concentration of acetic acid (20%) that
leads to PP7 disassembly as assessed by agarose gel electrophoresis of
virus-like particles. However, none of these resulted in a high yield
of active protein upon dialysis against 1 mM acetic acid
(as assessed by RNA-excess titrations), even when disulfide bonds were
reduced (results not shown). The method we eventually settled on uses
denaturation in 7 M urea in the presence of 10 mM DTT at pH 8.5 followed by dialysis against 1 mM acetic acid, 1 mM DTT. RNA-excess binding
curves show that this procedure results in recovery of about 25-50%
active protein, assuming the stoichiometry of one RNA binding site per
coat protein dimer typical of the other coat proteins characterized so
far. The RNA binding activity of these preparations is stable for at
least a few weeks when stored at 4 °C.
RNA Binding--
The concentration of active protein in a given
preparation was determined by RNA-excess titrations in TMK buffer at pH
8.5 (19). To determine appropriate conditions for binding reactions, protein-excess titrations were then performed with RNA held at about 10 pM under a variety of conditions. Specifically, pH was altered over a range from 6.2 to 8.5 using MoMK and TMK buffers (see
"Experimental Procedures"). KCl concentration was also varied from
40 mM to 1.0 M, and Mg2+ was
omitted from some reactions. None of these alterations had much effect
on binding, and most of the conditions we used gave a dissociation
constant close to 1 nM (Figs.
3 and
4). Binding curves generated in the
absence of Mg2+ were indistinguishable from those produced
in the presence of 10 mM magnesium acetate (data not
shown). The interaction was also essentially independent of pH over the
range we tested. Relatively large changes in KCl concentration also had
only modest effects on binding; Kd varied hardly at
all between 0 and 0.6 M. Even 1 M KCl resulted
in only a 4-fold elevation of Kd.
That RNA binding by PP7 coat protein in vitro is specific
for PP7 RNA was indicated by the failure of MS2 RNA to be tightly bound
(Fig. 5).
Mapping the RNA Binding Site of PP7 Coat Protein--
We
previously mapped amino acid residues contributing to the RNA binding
sites of MS2 and Q
In
In
In
The results of our mutational analyses are illustrated in Fig. 6, where
a map of residues whose substitution specifically affects RNA binding
is shown superimposed on a schematic of the coat protein Our experiments demonstrate that the RNA hairpin shown in Fig. 1
is indeed a target for binding by PP7 coat protein. It is a functional
translational operator in vivo (Table I) and is bound with a
Kd of about 1 nM in vitro
(Fig. 5). Thus, it seems that, like the other RNA phages, PP7 probably
utilizes coat protein to translationally repress replicase synthesis.
The structure of the PP7 operator differs significantly from those of
other RNA phages characterized previously, and neither the MS2 nor Q The protein-RNA interaction shows little dependence on salt
concentration over the range of 40 mM to 1 M,
suggesting that electrostatic interactions are relatively unimportant
in stabilizing the PP7 RNA-protein complex. This is a little
surprising, given that the RNA binding surface of PP7 coat protein has
four basic residues (three arginines and one lysine) and that
translational repression is sensitive to substitution at these sites.
Moreover, RNA binding by other coat proteins is sensitive to salt. For
example, MS2 coat protein, which utilizes five ion pair contacts in its complex with RNA, shows a significant dependence on salt concentration. Its Kd for operator RNA increases about 40-fold as
the KCl concentration approaches 1 M (19). GA and Q Changing pH over the range 6.2-8.5 also had little effect, indicating
that RNA binding is not substantially affected by protonation or
deprotonation of any groups with pKa values in this range. Other coat proteins so far characterized exhibit a variety of
behaviors with respect to pH. MS2 and GA have broad pH optima centered
around pH 7.0 and show only small changes in binding affinity between
pH 6.5 and pH 8.0 (19, 20). Above pH 8.0 the binding affinities of MS2
and GA begin to fall, behavior that has been attributed, at least in
the case of MS2, to the deprotonation of Tyr85 (22). In the
crystal structure of the MS2 RNA-protein complex, Tyr85 is
H-bonded through its phenolic hydroxyl to the phosphate of U-5. Substitution with phenylalanine abolishes this pH dependence. RNA
binding by Q Our mutational analyses provide a map of the PP7 coat protein RNA
binding site (Fig. 6). However, it should be noted that this could be
an incomplete map for reasons that include the following.
(i) Any amino acids that might contribute to the binding site but not
targeted for substitution are naturally omitted from this analysis. We
directed mutations to amino acids whose structural locations made them
likely candidates for RNA-contacting residues, and we cannot rule out
the possibility that other amino acids, for example those in the loops
that connect (ii) We also point out that substitution of a non-RNA-contacting
residue might sometimes cause a failure to bind RNA. For example, a
substitution may result in the introduction of a larger amino acid than
wild type, thus interfering sterically with RNA binding. However, at
only three positions (Ala52, Asp60, and
Val83) did we fail to isolate substitutions whose side
chains are smaller than the wild-type residue. It is also possible that
the substituted amino acid could alter the conformation of a true
RNA-contacting residue, affecting RNA binding secondarily. These
limitations are inherent in the approach utilized here and emphasize
the importance of structural studies as a companion to genetics and biochemistry.
(iii) Since our method includes a screen for assembly of ethidium
bromide stainable capsids, any amino acid whose substitution always
results in a folding or stability defect, or which causes failure to
encapsidate host RNAs, would be invisible to this analysis. However,
none of the sites we targeted on the coat protein surface failed to
yield at least one mutant that satisfied this requirement.
(iv) Any mutations that were to increase the nonspecific affinity of
coat protein for RNA might produce a repressor-defective phenotype by
increasing the competitive effects of host RNAs.
It is instructive to compare the binding site of PP7 with that of MS2,
whose complex with RNA is understood in great detail and was dealt with
in a recent review (23). Many of the amino acids that make up the two
sites are located in homologous positions on their respective
. Here we show that PP7 coat
protein is a specific RNA-binding protein, capable of repressing the
translation of sequences fused to the translation initiation region of
PP7 replicase. Its RNA binding activity is specific since it represses
the translational operator of PP7, but does not repress the operators
of the MS2 or Q
phages. Conditions for the purification of coat
protein and for the reconstitution of its RNA binding activity from
disaggregated virus-like particles were established. Its dissociation
constant for PP7 operator RNA in vitro was determined to be
about 1 nM. Using a genetic system in which coat protein
represses translation of a replicase-
-galactosidase fusion protein,
amino acid residues important for binding of PP7 RNA were identified.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet surface upon which the RNA is bound.
Each of the coat proteins uses a common structural framework to bind
different RNAs, thereby presenting an opportunity to investigate the
basis of specific RNA-protein recognition. In previous work we
characterized the RNA binding sites of MS2, GA, and Q
coat proteins
(8-10). Here we describe the RNA binding properties of the coat
protein of PP7, an RNA bacteriophage of Pseudomonas
aeroginosa whose coat protein shows only 13% amino acid
sequence identity to that of MS2. We present the following findings. 1)
The coat protein of PP7 is a translational repressor. 2) An RNA hairpin
containing the PP7 replicase translation initiation site is
specifically bound by PP7 coat protein both in vivo and
in vitro, indicating that this structure represents the
translational operator. 3) The RNA binding site resides on the coat
protein
-sheet. A map of this site is presented.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase under
translational control of PP7 coat protein. A plasmid we call pT7ropP7
contains the same EcoRI-BamHI operator sequence under control of the T7 promoter in pT7-1 (from U.S. Biochemical Corp.).
-32P]ATP by
in vitro runoff transcription of BamHI-cleaved
pT7ropP7, a plasmid that contains the PP7 operator sequence of Fig. 1
fused to the T7 promoter of pT7-1 (U.S. Biochemical Corp.). The
quantity of RNA retained after passage of the samples through the
nitrocellulose filter was determined using a Packard phosphorimager.
RNA-excess titrations of coat protein (at about 50 nM) gave
the concentration of active coat protein. Protein-excess titrations of
RNA at about 10 pM were used to determine
Kd. Binding reactions were performed under a variety
of conditions. To determine the pH dependence of RNA binding, buffers
in the pH range from 6.2 to 7.7 were made using 0.1 M MOPS
(MoMK buffers). In the pH range from 7.0 to 8.5, buffers contained 0.1 M Tris (TMK buffers). To measure ionic strength dependence
of binding, KCl was added to final concentrations ranging from 40 mM to 1.0 M. Magnesium ions were introduced
into most reactions by the addition of magnesium acetate to a
concentration of 10 mM. In our standard reactions, binding
was allowed to come to equilibrium during an incubation period of 60 min on ice. In an effort to determine whether equilibrium was attained
under these conditions, a series of protein-excess binding curves was produced after incubation for different time periods. Incubation times
ranging from 30 min to 8 h gave identical results.
-galactosidase from pRZ5, pRZP7, and pRZQ5 (10, 13).
Assays of
-galactosidase were performed using the method described
by Miller (16).
-D-galactoside (X-gal). After
24 h of growth at 37 °C, individual blue colonies were used to
inoculate 1-ml cultures in LB medium, which were grown to saturation overnight. To determine whether capsids were present, cells were harvested by centrifugation, resuspended in 0.25 ml of 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, and disrupted
by sonication. After removal of cellular debris by centrifugation,
glycerol and bromphenol blue were added and the samples were applied to
a 1% agarose gel containing 50 mM sodium phosphate, pH
7.4, 1 mM EDTA. Electrophoresis was conducted until the dye
had migrated 10 cm. The gel was stained with ethidium bromide, and
capsids were visualized by UV illumination. Mutants that passed this
capsid assembly test were subjected to DNA sequence analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
.
Therefore, our first task was to determine whether PP7 coat protein is
indeed a translational repressor and to identify its RNA binding
target. We had previously constructed two-plasmid systems for MS2, GA,
and Q
in which coat protein expressed from one plasmid represses
translation of a replicase-
-galactosidase fusion protein expressed
from a second plasmid (9, 10, 13). Inspection of the sequence of PP7
RNA reveals the potential RNA hairpin structure shown in Fig.
1. It contains the translational start of
the replicase gene. By analogy to the other phages, we felt this was
the best candidate for the PP7 translational operator.
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Fig. 1.
The sequences and secondary structures of
translational operators from MS2, Q , and
PP7.
-galactosidase gene, which lacks its own
initiator AUG. This plasmid, from a different incompatibility group
than pP7CT, expresses
-galactosidase from the lac
promoter and confers chloramphenicol resistance. If the sought-after
RNA-protein interaction exists, these manipulations should place
-galactosidase under translational control of PP7 coat protein in
E. coli cells harboring both plasmids. Repression would be
revealed by a failure to produce the blue color typical of
-galactosidase-producing colonies on X-gal plates.
two-plasmid systems, repression was weak. Attempts to
purify coat protein from cells bearing pP7CT revealed the probable
cause of poor repression; very little coat protein was being produced
(results not shown). Inspection of the PP7 sequence suggested that the
coat protein gene of this Pseudomonas phage possesses a
ribosome binding sequence that may be poorly adapted for translation
initiation in E. coli. Therefore, we attached the PP7 coat
protein coding sequence to a strong E. coli ribosome binding
sequence as follows. We PCR-amplified the coat sequence using a 5'
primer that introduced an NcoI site at the initiator AUG.
The 3' primer was the same as described above and introduced a
BamHI site downstream of the coat termination codon. The
insertion of this NcoI-BamHI fragment into pET3d
(12) attached the PP7 coat sequence to the T7 promoter and gene 10 ribosome binding site. The resulting plasmid, which we call pETP7CTNc, was introduced into E. coli strain BL21(DE3)/plysS where,
after induction with
isopropyl-1-thio-
-D-galactopyranoside, the PP7 coat
protein was copiously produced (results not shown). We note, parenthetically, that the NcoI mutation changes the identity
of the second amino acid in the coat sequence from serine to alanine. This change is predicted to result in the retention of the N-terminal methionine, which is normally removed by methionine aminopeptidase (18). However, as shown by the results that follow, these changes do
not seem to compromise the ability of the protein to carry out
translational repression or to assemble into a virus-like particle.
, a strain that contains none of the
apparatus for T7-based overexpression. Therefore, we excised the PP7
coat sequence, together with the upstream, gene 10-derived ribosome
binding sequence from pETP7CTNc by digestion with XbaI and
BamHI and inserted it into the corresponding sites of
pUC119, creating pP7CTNcXb. This plasmid produces increased amounts of
PP7 coat protein compared with pP7CT and efficiently represses
translation. Relative levels of
-galactosidase activity produced in
cells containing pRZP7 and either pP7CTNcXb or pUCter3 (a
non-coat-producing control) are shown in Table
I, where we see that PP7 coat protein
represses synthesis of the replicase-
-galactosidase fusion protein
to about 7% the level observed in the non-repressed control. The
repression is specific since other coat proteins (MS2 and Q
) do not
efficiently repress pRZP7, and because PP7 coat does not efficiently
repress the operators of these other phages (although it partially
represses the Q
operator).
Relative -galactosidase production by bacteria containing the
indicated plasmids
, and PP7 coat proteins, respectively. The plasmids called
pRZP7, pRZQ5, and pRZ5 contain the PP7, Q
, and MS2 operators fused
to lacZ. The values shown are percentages of the activities
found in the non-repressing (pUCter3) controls.
coat protein
(3). The FG loops of different coat protein dimers converge at the
3-fold (quasi-6-fold) and 5-fold symmetry axes in the virus particle,
where they participate in interactions that stabilize the capsid. If
disulfides are present, their reduction could be important to the
recovery of RNA binding activity during the renaturation step. To
determine the presence of interchain disulfides and to establish
conditions for their reduction, we conducted an experiment in which
virus-like particles, prepared by chromatography on Sepharose CL4B,
were exposed for 60 min to various concentrations of DTT at pH 8.5. The
reaction was terminated by the addition of excess
N-ethylmaleimide and applied to a polyacrylamide gel
containing SDS under non-reducing conditions. Fig.
2 shows that unreduced coat protein runs
as two bands, which probably correspond to cyclic pentamers and
hexamers. This interpretation is consistent with the formation of
disulfide bonds between polypeptide chains at the 5-fold and 3-fold
(quasi-6-fold) axes of the T = 3 icosahedral virus particle. These
species presumably take the form of 5-membered and 6-membered coat
protein rings because of the arrangement of coat proteins around the
symmetry axes. This pattern of disulfide bonding was observed
previously in Q
and, while this work was in progress, in the
structure of the PP7 phage itself (5-7). Partial reduction causes the
appearance of two new bands, which migrate slightly more slowly than
the completely unreduced cyclic pentamer/hexamer species. These
probably represent the linearization of the cyclic pentameric and
hexameric coat species by reduction of a single disulfide bond. Further
reduction results in the appearance of species whose molecular sizes
correspond to monomer, dimer, trimer, and tetramer. A DTT concentration
of 10 mM results in nearly complete reduction during the
60-min time course of the reaction.
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Fig. 2.
A, SDS-gel electrophoresis of the
products of partial reduction of interchain disulfide bonds in PP7
virus-like particles. Identifications of the various species are based
on the number and order of the bands and on the results shown in
B. B, plot of the mobilities of the partially
reduced PP7 coat proteins (open circles). The
molecular weights of putative monomer through hexamer species were
predicted from the monomer molecular weight of 14,000. The designations
c-5mer and c-6mer refer to the circular 5-mer and
6-mer species described in the text. The migration behaviors of four
molecular weight markers are shown for comparison (closed
circles). The markers were bovine serum albumin (68 kDa),
ovalbumin (43 kDa), chymotrypsinogen (25.7 kDa), and hen lysozyme (14 kDa).
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Fig. 3.
Dependence on pH of the dissociation constant
of PP7 coat protein for its translational operator.
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Fig. 4.
Dependence on KCl concentration of the
dissociation constant of PP7 coat protein for its translational
operator.
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Fig. 5.
A typical protein excess binding curve
conducted in MoMK buffer at 80 mM KCl. Open
squares represent the binding of PP7 coat protein to the PP7
operator. Closed circles are the binding by PP7 coat protein
of the MS2 operator RNA.
coat proteins by random mutagenesis, followed by
selection of mutants that failed to repress
-galactosidase synthesis
in the two-plasmid systems already described (9, 10, 13). A further
screen for capsid assembly eliminated mutants whose repressor defects
were consequences of failure to properly fold, and DNA sequence
analysis identified the affected amino acid residues. This procedure
resulted in the maps of the MS2 and Q
sites shown in Fig.
6. They use overlapping but non-identical sets of amino acids, and in each case the binding site resides on the
surface of the coat protein
-sheet. Because of the likelihood that
the RNA binding site of PP7 also resides on the
-sheet, we adopted a
site-directed mutational strategy to identify amino acids involved in
RNA binding. At the outset our only guide to PP7 coat protein structure
was its sequence homology with other coat proteins (14). On the basis
of amino acid sequence alignments with proteins of known
three-dimensional structure, we were able to identify amino acids
likely to reside within each of the
-strands. In
E and
G, the
alignments were sufficiently clear to allow us also to predict with
reasonable confidence those amino acids that reside on the surface of
the sheet and that therefore are potential RNA-contacting residues. We
targeted the codons for each of these amino acids for randomization
using the procedure given under "Experimental Procedures." In
F,
however, ambiguities in the sequence alignments made the assignment of
surface residues uncertain. Since the side chains of neighboring amino
acids project from opposite sides of the sheet, improper assignment of
the register of the
-strand could result in mistaken assignment of
the inside/outside arrangement of the amino acid side chains for the
entire strand. Therefore, in
F we randomized the codons for every
residue, reasoning that because substitutions oriented toward the
hydrophobic core are unlikely to exert any direct effect on RNA
binding, mutations that destroy translational repression by
substituting such residues probably exert their effects by disrupting
protein folding or stability. Mutants of this class should yield blue
colonies and will not produce virus-like particles. On the other hand,
residues whose substitution results in an RNA binding defect with
retention of proper folding (i.e. blue colonies with
production of capsids) must reside on the solvent-exposed surface of
the
-sheet where they can directly alter contacts with RNA. The
method we used for determining the presence of virus-like particles in
cell extracts is described under "Experimental Procedures," and the
results are shown in Fig. 7. The results
of mutational analysis, described in detail below, are summarized in
Table II and correlated with coat protein
structure in Fig. 6.
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Fig. 6.
A schematic representation of the structural
locations of amino acids substituted in repressor-defective,
assembly-competent mutants of MS2 (A),
Q (B), and PP7
(C).
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Fig. 7.
Agarose gel electrophoresis of capsids
produced by PP7 coat protein and various repressor-defective
mutants.
Translational repression by the PP7 repressor-defective mutants of
-galactosidase synthesis from pRZP7
E we mutagenized the codons for residues Arg39,
Thr41, Ser43, and Arg45. We found
that Arg39 and Arg45 yielded substitutions
resulting in the repressor-defective, assembly-competent phenotype,
thus implicating these amino acids in the RNA binding site. The two
other
E surface residues, Thr41 and Ser43,
never gave mutants in this class despite repeated efforts to isolate
them. All repressor-defective Thr41 and Ser43
mutants were also defective for assembly. However, sequence analysis of
some randomly chosen repressor-competent clones taken from Thr41 and Ser43 mutagenesis experiments
revealed that Thr41 can be replaced by arginine while
Ser43 tolerates substitution by alanine. Thus, the
identities of these residues are not crucial for RNA binding.
G residues, Val83, Ser85, and
Thr89 are identified as contributors to the RNA binding
site because substitution of these amino acids can result in the
repressor-defective, assembly-competent phenotype (although we should
note that no Ser85 substitutions gave normal capsid
yields). No repressor-defective mutants that satisfied the capsid
assembly criterion were found for Thr81 and
Asp87. However, sequence analysis of several
repressor-competent clones randomly selected from the mutant library
shows that RNA binding tolerates replacement of Thr81 by
serine, while Asp87 can be replaced by asparagine, serine,
or cysteine. Because of the close similarity of threonine and serine,
we cannot state with confidence that Thr85 is not a
constituent of the binding site.
F, where we mutagenized every amino acid, we never found an
assembly-competent repressor defect affecting any of the odd-numbered residues 51, 53, 55, 57, 59, or 63. On the other hand, we identified a
number of even-numbered residues in
F whose substitution resulted in
the repressor-defective, assembly-competent phenotype. These results
argue that the side chains of the odd-numbered amino acids of
F
reside in the core of the protein where they cannot contact RNA, thus
identifying the even-numbered amino acids as residing on the
solvent-exposed side of the sheet. This assignment has now been
verified in the x-ray structure of PP7 (5). Because they gave
repressor-defective, assembly-competent mutants, residues Ala52, Arg54, Lys58, and
Asp60 are identified as constituents of the RNA binding
site. Residues Asn56 and Ala62, although they
must reside on the RNA binding surface, did not yield
repressor-defective, assembly-competent mutants. Furthermore, sequence
analysis of randomly selected repressor-competent clones shows that
Asn56 tolerates substitution by lysine, and that
Ala62 tolerates threonine, arguing that they play no
crucial role in RNA binding.
-sheet.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
coat proteins efficiently repressed translation from the PP7 operator.
Moreover, PP7 coat protein showed little ability to repress the
operators of these other phages. Thus, the interaction is specific and
represents an example of the ability of the coat protein structural
framework to adapt by mutation to the binding of different RNAs. Future
studies will determine which features of RNA structure are necessary
for interaction with PP7 coat protein.
coat
proteins show similar dependences on salt concentration (20, 21).
coat protein also exhibits a broad optimum, but it is
centered around 6.0 and drops precipitously above pH 8 (21). Q
coat
protein has tyrosine at the position 85 equivalent, but the details of
its interactions with RNA are as yet unknown. However, this tyrosine
presumably participates in binding since substitution with histidine
reduces its affinity for RNA about 5-fold (10). PP7 contains valine at
the position 85 homologue.
-strands, or in
C or
D, might play a role in RNA binding.
-sheets. However, only 5 of the 15 amino acids on the surfaces of
the E, F, and G
-strands of the two proteins are identical (Table
III). Three of these are at positions 41, 43, and 58 (PP7 numbering), amino acids whose identities are highly
conserved among coat proteins generally. In MS2 it is easy to
understand their significance to the interaction with RNA; these amino
acids are involved in important interactions with A-10 and A-4 in the
translational operator. However, our results show that translational
repression by PP7 is tolerant of substitution of two of these residues,
Thr41 and Ser43. Given the striking divergence
of their RNA binding site sequence requirements, and considering the
obvious differences in their translational operators, it seems likely
that MS2 and PP7 use somewhat different modes of RNA binding.
Amino acids present on the surfaces of -strands E, F, and G of MS2
and PP7 coat proteins
Of course it is important to remember that the maps shown here are, in actuality, composites of two sites. The 2-fold symmetry of the dimer means that the RNA ligand may bind in either of two orientations. The makeup of a single, asymmetric site has already been determined for MS2 on the basis of heterodimer complementation experiments and by direct structural analysis of the RNA-protein complex, but similar information is not yet available for PP7.
It is striking that the coat protein-operator RNA interaction is conserved across a broad evolutionary spectrum of RNA phages. Thus, the translational repressor and its RNA target represent members of a co-evolving pair; mutations in one are compensated for by changes in the other so as to preserve the interaction. Clearly, retention of this RNA-protein interaction is important to these phages generally. This is also indicated by the observation that, although the coat protein-operator RNA interaction apparently is not absolutely required for virus viability, MS2 mutants with operator-inactivating mutations suffer a growth disadvantage compared with wild-type (24, 25). Possible explanations for the importance of this interaction include the following. (i) Replicase overproduction might reduce virus burst size by unnecessarily taxing the protein synthetic apparatus of the cell. (ii) The interaction may aid in recognition of viral RNA for selective encapsidation. (iii) Translational repression might serve to clear the replicase cistron of ribosomes, thereby facilitating genome replication or packaging. Any or all of these explanations may apply, as well as others we have not yet imagined.
The RNA phage coat proteins provide a structural platform upon which a
diversity of RNA structures can be bound. Although the translational
operators of the various RNA phages share some common features
(i.e. they are all stem-loop structures with bulged adenosines), the length of the stem, the size of the loop, and the
position and importance of the bulge vary. For at least one coat
protein-RNA interaction (Q), deletion of the bulge has only a modest
effect on binding, whereas, for others (MS2 and GA), the bulge is
essential. Although at present it is unknown how far the RNA binding
specificity of coat protein can be made to diverge from these natural
examples, we suggest that the coat protein
-sheet presents a surface
adaptable to the binding of many different RNAs, some of which could
differ substantially from any of the natural RNA targets so far
characterized. Work currently under way seeks to address this issue.
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FOOTNOTES |
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* This work was supported by a grant from the National Institutes of Health.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. Tel.:
505-272-0071; Fax: 505-272-9494; E-mail: dpeabody@salud.unm.edu.
Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M102411200
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ABBREVIATIONS |
---|
The abbreviations used are:
PCR, polymerase
chain reaction;
DTT, dithiothreitol;
MOPS, 4-morpholinepropanesulfonic
acid;
X-gal, 5- bromo-4-chloro-3-indolyl--D-galactoside.
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REFERENCES |
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