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INTRODUCTION |
The double-stranded DNA-gene product 3 (DNA-gp3)1 complex of the
Bacillus subtilis bacteriophage
29 is packaged
efficiently into a prohead with the aid of the ATPase gp16 and ATP
hydrolysis in a completely defined in vitro system (1). A
unique 174-base
29-encoded RNA, termed prohead RNA (pRNA), is
present on the portal vertex (head-tail connector) of the prohead and
is an essential constituent of the DNA packaging machine (2). pRNA is
hypothesized to bind a supercoiled DNA-gp3-gp16 complex to link the DNA
and prohead (3), recognize the left end of the DNA-gp3, which is packaged first (4), and unite with gp16 to form the DNA translocating ATPase (5).
The secondary structure of the pRNA was established by a phylogenetic
analysis (6). pRNA binding to proheads is specific, rapid, and
irreversible in the presence of 10 mM Mg2+.
Proheads protect nucleotides 22-84, 5
to 3
, of pRNA from
ribonuclease attack, and the use of site-directed mutants of pRNA has
identified elements and sequences of pRNA that are required for prohead
binding and DNA-gp3 packaging (7-9). The mutant studies also
identified a pseudoknot involving a nine-membered bulge loop and a
five-base hairpin loop in pRNA that is essential in DNA-gp3 packaging
(9). Recently, the pseudoknot has been shown to be an intermolecular interaction, requiring just two base pairs, that links six identical molecules of pRNA into a structure that is positioned on the portal vertex of the prohead.2
The structure of pRNA as it interacts with the proteins of the
packaging machine and DNA-gp3 is integral to understanding the
mechanism of DNA packaging. To better define the sequence and
structural elements of pRNA essential for prohead binding, a 62-base
segment of the prohead binding domain (residues 30-91) was partially
randomized, and pRNA aptamers, high affinity ligands that bind the
prohead, were selected in vitro from a large pool of RNA
molecules. Subsequently, 23 bases of this domain (residues 45-62 and
81-85) that include the intermolecular pseudoknot were completely
randomized in a second in vitro selection analysis that was
based on prohead binding and included assays of DNA-gp3 packaging. The
results demonstrated more precisely the pRNA sequences and elements
essential for prohead binding and DNA-gp3 packaging and identified the
intermolecular base pairing required for pRNA oligomerization. In
general, the selections recapitulated the natural evolution of
pRNA.
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EXPERIMENTAL PROCEDURES |
Construction of RNA Libraries--
The in vitro
selections of pRNA aptamers that bind the prohead were patterned after
the method termed systematic evolution of ligands by exponential
enrichment (SELEX) (10), which utilizes variation, selection, and
replication. A vast repertoire of RNA molecules produced from a
randomized template are bound to a target, selected molecules are
amplified as DNA that is competent for in vitro
transcription, and the newly transcribed RNAs, enriched for better
binding sequences, are subjected to selection to begin the next
cycle.
The DNA oligonucleotides used as templates in initial transcription for
SELEX experiments I and II were purchased from Oligos Etc. Inc. In
SELEX I, the randomized region of the template was based on positions
30-91 of the wild-type sequence (see Fig. 1, A and
C) with a 16% mutation rate. The 110-nucleotide DNA
template for the original RNA pool, with degenerate nucleotides in the 62-base prohead binding domain represented by N, was as follows: 5
-GGGCCCTTTG TCATGNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN
NNNNNNNNNN NNNNNNNNNN NNNNNNNACA TGACAAAGGG
CCC*TATAGTGAGTCGTATTA-3
(the transcription start site
is indicated with an asterisk, and the T7 promoter is underlined).
Primer 1 (5
-GC*TCTAGATAATACGACTCACTATAGGGCCCTTTGTCATGT-3
(the XbaI site is indicated with an asterisk, and the
T7 promoter is underlined)) was used for making the double-stranded DNA
template and for PCR amplification. Primer 2 (5
-AACTGCA*GGGCCCTTTGTCATG-3
(the PstI site is
indicated with an asterisk)) was used for reverse transcription and PCR
amplification.
The 120-nucleotide template for the original RNA pool of SELEX II in
the complete randomization of pRNA residues A45 through
G62 and U81-U85 (see Fig. 1,
A and D), with randomized nucleotide positions
represented by N, was as follows: 5
-TTAGGAAAGT AGCGTGCACT
TTTGCCATGA TTGACNNNNN ATCAACAAAG TATGTGGGNN NNNNNNNNNN NNNNNNTAAT
CCCCAACATA CACATGACAA TGGAAGTACC GTACCATTCC-3
. Primer 1 (5
-CCGG*AATTCTAATACGACTCACTATA*GGAATGGTACGGTACTT-3
(the EcoRI site and the transcription start site are
indicated with an asterisk, and the T7 promoter is underlined)) was
used for making the double-stranded DNA template and for PCR
amplification. Primer 2 (5
-CGCG*GATCC*TTAGGAAAGTAGCGTGC-3
(the
BamHI and DdeI sites are indicated with an
asterisk)) was used for reverse transcription and PCR
amplification.
The 110-base single-stranded DNA template (SELEX I) and the 120-base
single-stranded DNA template (SELEX II) were annealed to their
respective primer-2 oligonucleotides and extended to form
double-stranded DNA with five cycles of PCR. The double-stranded DNA
templates in SELEX I and II were transcribed to produce original RNA
pools with complexities of 2.5 × 1014 and 7 × 1013 molecules, respectively. T7 transcription was carried
out as described (7) in a volume of 800 µl. T7 RNA polymerase was purchased from Ambion, Inc., and the reaction buffer was that of Life
Technologies, Inc.
In Vitro Selections--
pRNA-free proheads were produced by
isopropyl-1-thio-
-D-galactopyranoside induction of
Escherichia coli strain HMS174(DE3) (pAR7-8-8.5-10) as
described (7, 8). The binding affinity of proheads to pRNA was titrated
by the use of 32P-labeled wild-type pRNA (8, 9). The ratio
of molecules of RNA to proheads was 100. For each selection, 200 µg
of proheads (approximately 1013) were used. Competitive
binding was performed in 400 µl of TM (0.05 M Tris-HCl
(pH 7.8), 0.01 M MgCl2) buffer with 180 µg of yeast tRNA at room temperature for 30 min. RNA bound to proheads was
separated from free RNA by centrifugation in a 5-20% sucrose gradient
in the Beckman SW55 rotor at 35,000 rpm for 30 min at 4 °C. The
prohead band was isolated, and 1 volume of H2O was added to
dilute the sucrose. RNA was recovered by phenol extraction and ethanol
precipitation in the presence of 0.5 M NH4AC.
cDNA was produced by SuperscriptTM RNase
H
reverse transcriptase (Life Technologies, Inc.) and
amplified by 30 cycles of PCR in four 100-µl reactions, and the DNA
was transcribed to start another round of selection. For reverse
transcription, the selected RNA was annealed to 1 µg of primer 2 by
heating in water for 3 min at 100 °C and then cooling the sample in
ice water. The reverse transcriptase was from Life Technologies, Inc.,
and reverse transcription was carried out in 40 µl as recommended by
the manufacturer. PCR cycle conditions, adapted from Longel et
al. (11), were as follows: 4 min at 94 °C for initial
denaturation, 1 min at 94 °C, 2 min at 45 °C, 3 min at 72 °C,
and a final 7 min at 72 °C. Wild-type pRNA was transcribed from
plasmid PRT71 digested by DdeI as described (7, 8). DNAs
from different cycles were cloned into pUC19 at XbaI and
PstI sites (SELEX I) or EcoRI and
BamHI sites (SELEX II).
Prohead Binding Assay--
The ability of RNA pools from each
selection to compete with wild-type pRNA for prohead binding was used
as a measure of relative binding affinity. The competition binding
assay was carried out as described (8, 9). Briefly, 1.4 pmol of
120-residue wild-type [32P]pRNA was mixed with 14 pmol of
an unlabeled RNA from each selection pool and 60 pmol of tRNA. 0.35 pmol of proheads were added to the RNA, and the fraction of
[32P]pRNA bound was measured by filtration. A competitor
activity for each RNA in the competition assay was derived by fitting
values for the fraction of wild-type [32P]pRNA bound to
the standard curve described by
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(Eq. 1)
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and solving for C, where Bf is the
fraction bound and C is the moles of competitor RNA per mole
of [32P]pRNA. Values were normalized to the no competitor
control (C = 0) and the wild-type pRNA competitor
control (C = 100) to give
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(Eq. 2)
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where Cn compares the competitor activity of
the test RNA to the wild-type pRNA, B10 is the
fraction of [32P]pRNA bound when a 10-fold excess of
wild-type pRNA is used as competitor, B0 is the
fraction of [32P]pRNA bound with no competitor RNA, and
Bt is the fraction of [32P]pRNA bound
when a 10-fold excess of test competitor is added.
Cloning and Sequencing of Variants--
DNA amplified from
different cycles of selection was cloned into the PstI and
XbaI sites of pUC19 and transformed into E. coli
strain DH5
. Clones with inserts were identified by negative
complementation. The plasmids were sequenced with Sequenase 2.0 (United
States Biochemical) using a protocol adapted from that provided by the
manufacturer.
DNA Packaging Assays--
In vitro DNA-gp3 packaging
was carried out as described (8, 12). Briefly, wild-type or mutant
pRNAs were bound to RNA-free proheads, and the reconstituted proheads
were then added to DNA-gp3, gp16, and ATP. After 30 min at
25 °C, unpackaged DNA was digested with DNase I, EDTA was added, and
packaged DNA was extracted from filled heads and quantified by agarose
gel electrophoresis.
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RESULTS AND DISCUSSION |
Structure of pRNA and Rationale for in Vitro Selection of pRNA
Aptamers for Prohead Binding--
Fig.
1A shows the proposed
secondary structure of the 120-base form of the 174-residue pRNA (6)
that is necessary and sufficient for DNA-gp3 packaging in the defined
in vitro system (2, 12). The shaded region that
marks nucleotides 22-84 is the footprint of proheads on pRNA generated
with the ribonucleases A, T1, and V1 (7). Analyses utilizing
oligonucleotide-directed mutant pRNAs show that both the sequence and
the secondary structure of residues 40-91 are important for prohead
binding and that elements of the A helix formed from residues 1-28 and
92-117 are needed for DNA packaging functions other than prohead
binding (8, 13). The secondary and tertiary structures of the prohead
binding region of pRNA have been probed by measuring binding of mutant pRNAs to RNA-free proheads and in vitro packaging of
DNA-gp3. A truncated pRNA of 62 residues (residues 30-91) retains
prohead binding activity but is insufficient for DNA-gp3 packaging
(9).

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Fig. 1.
Secondary structure of pRNA, mutant pRNAs,
and RNAs upon which templates for SELEX experiments were based.
A, secondary structure of the 120-base form of wild-type
pRNA (6). The helices are designated A, C, D, and
E. Shading shows the RNase footprint of the
prohead on pRNA (7). The boxes and line identify
a pseudoknot, helix G (9), that was recently shown to be an
intermolecular interaction that links identical molecules of pRNA into
dimers and hexamers needed for DNA-gp3 packaging in
vitro.2 B, compensatory changes to test the
G helix interactions shown in A. C, RNA structure
upon which the template for the original RNA pool of SELEX I was based.
Shading shows the 62-nucleotide domain (nucleotides 30-91)
that was partially randomized with a mutation rate of 16%.
D, pRNA that served as the basis for the template for the
original RNA pool of SELEX II. Shading marks residues 45-62
and 81-85, which were completely randomized.
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A pseudoknot in pRNA inferred from the phylogenetic studies was
confirmed by constructing mutants to change positions
45AACC48 to GCGA (mutant F6),
82GGUU85 to UCGC (mutant F7), or
45AACC48 to GCGA and
82GGUU85 to UCGC (mutant F6/F7) to restore the
hypothetical base pairing of the G helix (9) (Fig. 1B).
Neither F6 nor F7 pRNAs could reconstitute proheads for in
vitro DNA-gp3 packaging, whereas the F6/F7 double mutant pRNA was
as effective as wild-type pRNA in reconstituting proheads for DNA
packaging. Moreover, proheads reconstituted with either F6 or F7 pRNA
could not promote in vitro phage assembly in a
prohead-defective extract, whereas the F6/F7 pRNA was as effective as
wild-type pRNA in reconstituting proheads for phage assembly. Recently,
a mixture of F6 and F7 pRNAs was found to be as active in DNA-gp3
packaging as the double mutant F6/F7 and wild-type pRNA, showing that
the pseudoknot is intermolecular rather than intramolecular. This
intermolecular base pairing results in the formation of an oligomer
from six identical molecules of pRNA that is essential for efficient
DNA-gp3 packaging.2 To define the specific nucleotides of
pRNA needed for prohead binding and formation of the intermolecular
pseudoknot, segments of pRNA were partially or completely randomized,
and RNA aptamers for prohead binding were obtained by multiple rounds
of in vitro selection.
Partial Randomization of the Prohead Binding Domain of
pRNA--
The 62-base prohead binding domain (residues 30-91) shown
as the shaded region in the template of Fig. 1C
was partially randomized with a mutation rate of 16% as described
under "Experimental Procedures." The DNA template utilized the T7
promoter, and an initial pool of RNA molecules with a complexity of
5 × 1014 molecules was produced. RNA pools were
obtained from seven rounds of in vitro selection by prohead
binding and separated from unbound RNA by isolation of the prohead-pRNA
complexes in sucrose density gradients. cDNA was produced,
amplified by PCR, cloned, and sequenced.
The ability of pools of RNA from the various rounds of in
vitro selection to compete with the binding of
[32P]wild-type pRNA to proheads is shown in Fig.
2. From the fifth round, RNA pools were
equivalent to wild-type pRNA as competitors of prohead binding. tRNA
was used as a negative control for competition. RNAs sequenced from the
second and third rounds of selection showed changes at practically
every position of the prohead binding domain (not shown). On the
contrary, in the fifth round, changes in residues 46-48 of the C-E
loop and 81-84 of the D loop, which are involved in intermolecular
base pairings that produce dimers and hexamers of pRNA,2
were infrequent in 93 sequences, and most of the residues of the E stem
and residues 53-56 of the E loop were wild-type (Fig. 3 and Table
I). Covariation was detected within
individual sequences in six of nine base pairs of the C helix and all
six base pairs of the D helix (Fig. 3), lending strong support to the
model of pRNA secondary structure (6).

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Fig. 2.
Competition filter binding with RNA pools of
SELEX I. Competition binding assays were performed as described
under "Experimental Procedures." tRNA was used as a control.
Amounts of [32P]pRNA bound were as follows: input, 8529 cpm; no proheads, 224 cpm. The competition activities, which normalize
the [32P] pRNA fraction bound with wild-type pRNA
competitor (100%) and no competitor (0%) (8, 9), were as follows:
tRNA, 3%; pRNA, 100%; cycle 0, 4%; cycle 1, 8%; cycle 2, 12%;
cycle 3, 29%; cycle 4, 76%; cycle 5, 96%; cycle 6, 93%; and cycle
7, 97%.
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Fig. 3.
Sequences of 93 pRNA clones from selection
cycle five of SELEX I after partial randomization of the 62-nucleotide
prohead binding domain. The 62-base wild-type sequence is shown
above the mutant sequences. Numbers under the sequence are
the positions in pRNA. Capital letters above the wild-type
sequence designate the helices and loops of pRNA. Deviations from the
wild-type nucleotide at each position are given, and conservation of
the wild-type base is indicated with a dash.
Circled bases designate covariation within helices to
maintain base pairing.
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Table I
Summary of base changes at each position of the partially randomized
62-base prohead binding domain of pRNA in SELEX I
The 93 sequences are given in Fig. 3. Bases in boldface type are
wild-type, and the number before each base is the position in the
molecule. Intermolecular base pairing between residues 47,48CC and
83,82GG is necessary and sufficient for formation of dimers and
hexamers of pRNA.2 Most of the RNA sequences are wild-type
at these positions and at 46A and 84U which may contribute to the
intermolecular interaction. Also, changes were rare in three of four
base pairs of the E helix (residues 49C, 50U, and 51G, which pair with
62G, 61A, and 60C, respectively) and in the residues 53U, 54U, 55G, and
56A of the E loop.
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The data were consistent with previous results from site-directed
mutagenesis that demonstrated that the sequence and structure of the
C-E loop, the E stem and loop, and the D loop were of particular importance in prohead binding and DNA-gp3 packaging (8). The 62-base
pRNA is not active in DNA-gp3 packaging, and therefore, this study was
limited to prohead binding.
Complete Randomization of a Part of the CE Loop, the E Stem and
Loop, and the D Loop--
A second SELEX experiment focused on the
sequences and elements of pRNA crucial for prohead binding and produced
the entire 120-base pRNA needed for DNA-gp3 packaging assays.
Twenty-three bases (residues 45-62 and 81-85) (Fig. 1D)
were completely randomized to yield an initial RNA pool with an
expected complexity of 7 × 1013 molecules. RNA was
selected by multiple rounds of binding to proheads. cDNA was
produced, amplified by PCR, cloned, and sequenced. From the fifth
round, RNA pools were equivalent to wild-type pRNA as competitors of
prohead binding (Fig. 4). A sequencing
gel of pRNA pools from the initial through the fifth round of selection is shown in Fig. 5. The progression from
a population of random sequences to a population dominated by the
wild-type is dramatic. From cycle three, only 2 of 18 clones were
wild-type. From cycle four, 74 of 90 clones (82%) were wild-type, and
from cycle 5, 190 of 226 clones (84%)
were wild-type. Representative mutant sequences of the randomized regions from cycles 4 and 5 are given in
Tables II and III, respectively.

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Fig. 4.
Competition filter binding with RNA pools of
SELEX II. Competition binding assays were performed as described
under "Experimental Procedures." 5S rRNA was used as a control.
Amounts of [32P]pRNA bound were as follows: input, 14964 cpm; no proheads, 142 cpm. The competition activities, which normalize
the [32P]pRNA fraction bound with wild-type pRNA
competitor (100%) and no competitor (0%) (8, 9), were as follows: 5S
rRNA, 2%; pRNA, 100%; cycle 0, 4%; cycle 1, 12%; cycle 2, 16%;
cycle 3, 46%; cycle 4, 83%; cycle 5, 97%; and cycle 6, 91%.
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Fig. 5.
Sequencing gel of pools of RNA from the
initial selection through the fifth cycle of SELEX II.
Numbers above the lanes refer to the selection
cycles, and numbers at the left refer to positions in pRNA.
Emergence of the wild-type is clear in selection cycle 3.
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Table II
Representative mutant sequences of cycle 4 of SELEX II
Residues 45-62 and 81-85 were completely randomized, and pRNAs from
selection cycle 4 were sequenced. Conservation of the wild-type base is
indicated with a dash. Residues 47,48CC form the intermolecular
pseudoknot with 83,82GG. Circled bases show covariations.
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Table III
Representative mutant sequences of cycle 5 of SELIX II
Residues 45-62 and 81-85 were completely randomized, and pRNAs from
selection cycle 5 were sequenced. Conservation of the wild-type base is
indicated with a dash. Residues 47,48CC form the intermolecular
pseudoknot with 83,82GG. Circled bases show covariations. An
asterisk denotes the number of clones sharing the same
sequence.
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Tables II and III illustrate that residues 45-62 are wild-type in 22 of 31 sequences. On the contrary, changes occurred in residues 81-85
in all but three of the mutant pRNAs. The base 45-62 interval includes
the C-E bulge loop and the E stem and loop. Bases 81-85 make up the D
loop, residues of which form base pairs intermolecularly with the C-E
loop in pRNA oligomerization (addressed below). Substantial change in
the D loop shows that prohead binding per se does not
require pseudoknot formation, i.e. pseudoknot formation may
follow prohead binding. Moreover, mutant pRNA F6, which cannot form the
pseudoknot, still has 20% prohead binding competitor activity relative
to wild-type pRNA (9). The results suggest that the intermolecular
pseudoknot is not important for the initial interaction of pRNA with
the prohead but is important for subsequent interactions critical to
DNA-gp3 packaging activity.
The nine sequences of residues 45-62 that were not wild-type showed
abundant change (Tables II and III). Eight of these sequences showed
covariation in base pairs of the E helix, suggesting that maintenance
of this element is important in prohead binding. In the mutant pRNAs
4-1 (Table II) and 5-1, 5-14, and 5-15 (Table III), covariation
occurred in two of four base pairs of the E stem, and in the mutant
4-10 (Table II), covariation occurred in all four bases of the E
stem.
Folding of pRNA mutant sequences was predicted by the computer
algorithm MFOLD (14). Minor, even single, base changes in the pRNA
sequence can produce drastic alterations in the predicted foldings
(15). Studies on pRNA mutants with limited numbers of G to A or C to U
changes generated by bisulfite mutagenesis showed that the foldings of
pRNA have prognostic value. In each case in which the overall predicted
folding of a mutant differed substantially from the wild-type model,
production of RNA was not detected in vivo from the cloned
genes, presumably because of degradation of misfolded molecules by
endogenous ribonucleases. All 22 of the pRNA sequences of Tables II and
III that are wild-type for residues 45-62 were predicted to fold as
the model did (Fig. 1A), regardless of the D loop sequence.
This suggests that all major determinants of secondary structure are in
place in these mutants, and even changing all five bases of the D loop
did not perturb the predicted folding. However, only 6 of these 22 mutant pRNAs were active in DNA-gp3 packaging, in all cases because the residues 82GG83 were retained in the D loop,
providing the capability to form the intermolecular base pairs needed
for oligomerization (see below). None of the pRNAs with changes in the
segment that included residues 45-62 could reconstitute RNA-free
proheads for DNA-gp3 packaging in vitro.
The SELEX experiments defined the minimal requirements for
intermolecular base pairing leading to formation of pRNA dimers and
hexamers required for DNA-gp3 packaging.2 Previous work
suggested that the pseudoknot was a four-base pair interaction
involving nucleotides 45-48 and 85-82 (9) (see Fig. 1, A
and B). Full DNA-gp3 packaging activity was obtained with
the mutants 4-2, 4-5, 4-6, 5-6, 5-8, and 5-12, in which bases were
changed from U85 to C; U85 to G;
84UU85 to CA; U85 to C;
U81 to A; and 84UU85 to GA,
respectively (Tables II and III); thus, potential base pairing
involving U residues at positions 84 and 85 was not needed for
biological activity. The DNA-gp3 packaging results from SELEX II are
shown in Fig. 6. All of the biologically
active pRNAs maintained G at positions 82 and 83. A change of
G82 to C in mutants 4-3, 5-9 and 5-11; to A in mutants 4-8 and 4-11; and to U in mutants 5-10 and 5-18 while maintaining G at
position 83 resulted in loss of DNA-gp3 packaging activity. Similarly, a change of G83 to U in mutants 4-9 and 5-5 while
G82 was maintained resulted in loss of DNA-gp3 packaging
activity. As expected, when both G82 and G83
were changed to CC in mutants 4-4, 5-4, 5-7, and 5-16; UU in mutants
4-7, 5-2, and 5-19; AU in mutant 5-1; CA in mutants 5-3 and 5-13; AA in
mutant 5-15; and AC in mutants 4-1 and 5-20, DNA-gp3 packaging was not
observed. These results showed that base pairing between
47CC48 and 83GG82 is
necessary and sufficient for DNA-gp3 packaging. Data showing that this
intermolecular interaction produces a hexameric oligomer of pRNA will
be presented.2 Site-directed mutagenesis of these residues
has confirmed the results presented here and demonstrated that pRNAs
with a change of the wild-type bases
47CC48-83GG82 to
GC-CG, CG-GC, CU-GA, UC-AG, or GA-CU retain efficient DNA-gp3 packaging activity.2 Mg2+ is needed for
formation of dimers and hexamers of pRNA in solution. Dimers of pRNA
have been detected by native polyacrylamide gel electrophoresis at room
temperature, and dimers and hexamers were detected at 4 °C; proof of
the dimeric and hexameric forms of pRNA in solution was obtained by
analytical ultracentrifugation.2

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Fig. 6.
In vitro DNA-gp3 packaging activity of
proheads reconstituted with mutant pRNAs from selection cycle five of
SELEX II. DNA-gp3 packaged in the defined in vitro
system by RNA-free proheads reconstituted with pRNAs was extracted and
quantified following agarose gel electrophoresis. Shown is the amount
of DNA-gp3 added to each reaction (lane 1), DNA-gp3 packaged
by RNA-free proheads (lane 2), DNA-gp3 packaged by proheads
with wild-type (wt) pRNA (lane 3); and DNA-gp3
packaged by proheads reconstituted with the indicated mutant pRNAs
(lanes 4-23). Lanes from two gels were spliced with Adobe
Photoshop 4.0.
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Conclusions and Perspective--
The SELEX experiments
recapitulated the natural evolution of the C-E bulge loop and the E
stem and loop of pRNA. No alternatives to wild-type pRNA in terms of
the sequence and secondary structure of these elements were identified
by the SELEX experiments. The only analogues of wild-type pRNA with
apparently identical structure and function in DNA-gp3 packaging were
the mutants 4-2 (5-6), 4-5, 4-6, 5-8, and 5-12, which had D loop
changes of U85 to C, U85 to G,
84UU85 to CA, U81 to A, and
84UU85 to GA, respectively (Tables II and III);
all of these RNAs maintain 83GG82, which forms
intermolecular base pairs with 47CC48 of the
C-E bulge loop that are needed for DNA-gp3 packaging.2
Thus, the D loop functions primarily in intermolecular base pairing of
pRNA rather than prohead binding; however, deletion of both the D stem
and loop results in loss of prohead binding competitor activity
(8).
When the E stem and loop were wild-type in SELEX II sequences, the C-E
loop was invariably wild-type, whereas the D loop sequences showed many
changes (Tables II and III). Thus, the C-E loop must have prohead
binding function unrelated to pseudoknot formation, and the pseudoknot
is not a determinant of prohead binding. However, the potential for
base pairing of the D loop and the C-E loop that produces pRNA
oligomers was present in all SELEX II sequences active in DNA-gp3
packaging (Tables II and III), confirming studies with site-directed
mutants.2 Surprisingly, covariation in bases of the base
45-48 and 81-85 segments that form pseudoknot pairs was common in
sequences of cycle 3 of SELEX II that showed many changes in the base
45-62 and 81-85 intervals (data not shown); thus, pseudoknot
formation, although not required for prohead binding, may aid prohead
binding prior to evolution of a wild-type E stem and loop.
The nine-membered C-E bulge loop, the four-base pair E stem, and the
six-residue E loop constitute the crux of the prohead binding domain.
Deletion of the C-E loop results in loss of prohead binding competitor
activity (8). Inversion of the E stem residues 49CUGA52 and the complementary residues
62GACU59 to AGUC and UCAG, respectively,
results in prohead binding competitor activity of only 11% and no
detectable DNA-gp3 packaging activity, even though the pRNA was
predicted to fold as the wild-type did (8). Very few changes were
observed in these residues in SELEX I (Table I), and no changes were
observed in the G51-C60 pair. Covariation
occurred in the E stem in eight of nine mutants with multiple changes
in the residue 45-62 segment in SELEX II (Tables II and III). The
composite results show the importance of the E stem in both prohead
binding and DNA-gp3 packaging. Considering the E loop, no changes were
observed in residues U53, U54, and
A56 in SELEX I, and G55 was replaced only with
A (Table I). Previously, the highly conserved residues U54
and A56 were changed to A and C, respectively. Modest
decreases were observed in prohead binding competitor activity and a
4-fold and 2-fold drop, respectively, in capacity to reconstitute
proheads and support phage assembly in prohead-defective extracts (15). The SELEX I results suggested that the least important residues of the
E loop in prohead binding were G57 and U58, in
which change was quite frequent (Table I). Change of the four residues
54UGAG57 to CUUU resulted in drastic loss of
prohead binding competitor activity (8), underscoring the importance of
the E loop sequence.
The A helix of pRNA (see Fig. 1A) may be the principal site
of interaction with gp16 that results in formation of the unique RNA-dependent DNA translocating ATPase (5, 13). The A helix has been partially randomized with a mutation rate of 40%, and the
selection and sequencing of pRNA aptamers that bind gp16 is in
progress. Other goals include investigation of the role(s) of
oligomerization of pRNA in DNA-gp3 packaging, visualization of
pRNA hexamers on the prohead portal vertex, and determination of the
higher order structure of pRNA as it interacts with proteins of the DNA
packaging machine and the packaging substrate DNA-gp3.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X05973.