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Originally published In Press as doi:10.1074/jbc.M100045200 on May 22, 2001
J. Biol. Chem., Vol. 276, Issue 35, 32575-32584, August 31, 2001
Three-dimensional Interaction of Phi29 pRNA Dimer Probed by
Chemical Modification Interference, Cryo-AFM, and Cross-linking*
Yahya
Mat-Arip §¶,
Kyle
Garver¶ ,
Chaoping
Chen§§,
Sitong
Sheng,
Zhifeng
Shao, and
Peixuan
Guo**
From the Department of Pathobiology, Purdue
University, West Lafayette, Indiana 47907 and the
Department of Molecular Physiology and
Biological Physics, University of Virginia,
Charlottesville, Virginia 22908
Received for publication, January 3, 2001, and in revised form, May 17, 2001
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ABSTRACT |
Six pRNAs (p for packaging) of bacterial virus
phi29 form a hexamer complex that is an essential component of the
viral DNA translocating motor. Dimers, the building block of pRNA
hexamer, assemble in the order of dimer  tetramer hexamer. The
two-dimensional structure of the pRNA monomer has been investigated
extensively; however, the three-dimensional structure concerning the
distance constraints of the three stems and loops are unknown. In this report, we probed the three-dimensional structure of pRNA monomer and
dimer by photo affinity cross-linking with azidophenacyl. Bases 75-81
of the left stem were found to be oriented toward the head loop and
proximate to bases 26-31 in a parallel orientation. Chemical
modification interference indicates the involvement of bases 45-71 and
82-91 in dimer formation. Dimer was formed via hand-in-hand contact, a
novel RNA dimerization that in some aspects is similar to the kissing
loops of the human immunodeficiency virus. The covalently linked
dimers were found to be biologically active. Both the native dimer and
the covalently linked dimer were found by cryo-atomic force microscopy
to be similar in global conformation and size.
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INTRODUCTION |
Interactions between RNA molecules play diverse roles in different
biological systems. Dimerization of retrovirus RNAs via kissing loops
is believed to govern essential steps in the retroviral life cycle,
including translation, reverse transcription, RNA encapsidation, and
virion assembly (1, 2). During the early events of pre-mRNA
splicing, there are several types of interactions through a network of
RNA-RNA, RNA-protein, and protein-protein contacts (3-6). In addition,
RNA-RNA interactions are also involved in the cleavage of tRNA by RNase
P (7-9), and in genetic regulations in bacteria (10, 11), eukaryotes
(12), plants (13), mammals (14), and plasmids (15).
An intermediate step in morphogenesis of phi29, a bacterial virus that
infects Bacillus subtilis, is the formation of a DNA-filled capsid generated through the translocation of genomic dsDNA into an
empty capsid shell (procapsid or prohead), a process called DNA
packaging (for reviews, see Ref. 16). Translocation of
double-stranded DNA into the procapsid requires a pair of
noncapsid proteins and a virus-encoded RNA (17, 18), called
pRNA1 (p for packaging). The
120-base pRNA participates in the DNA packaging reaction but is not a
part of the mature phi29 virion. The pRNA binds to the connector (the
unique site where DNA goes through) of procapsids in the presence of
Mg2+ (19). The pRNA also appears to be directly involved in
the DNA translocation process and leaves the procapsid after DNA
packaging is completed (20).
To elucidate the role of the pRNA in this DNA translocating motor, it
is crucial to know how many copies of the pRNA are involved in each DNA
packaging event. We have developed three approaches to determine the
stoichiometry of the pRNA. These three approaches have led to the
conclusion that six pRNAs are required for the function of each motor.
The first determination of pRNA stoichiometry involved the
use of binomial distribution (21, 22). pRNAs with mutations in the
5'/3' paired region (the DNA translocation domain) retained procapsid
binding capacity but failed to package DNA. When mutant pRNA and
wild-type pRNA were mixed at various ratios in in vitro assembly assays, the probability of procapsids that possess a certain
amount of mutant and a certain amount of wild-type pRNA was determined
by the expansion of a binomial (p + q)Z,
where Z is the total number of pRNA per procapsid, and
p and q represent the percent of mutant and
wild-type pRNA, respectively, used in reaction mixtures. For example,
if we assume that Z is 3, the probability of all
combinations of mutant and wild-type pRNAs on a given procapsid can be
predicted by the expansion of the binomial: (p + q)3 = p3 + 3p2q + 3pq2 + q3 = 100%. The yield of virions from empirical
data was plotted and compared with a series of predicted curves to find
a best fit. Our results showed that approximately five to six pRNAs
were needed for each procapsid to package DNA, explaining the high inhibition efficiency of mutant pRNA (23).
The second approach for stoichiometry determination utilized
serial dilution factor of pRNA versus the yield of virions
assembled in vitro (21). The larger the stoichiometry of the
component, the more dramatic the influence of the dilution factor on
the reaction. A slope of one indicates that one copy of the component is involved in the assembly of one virion. A slope larger than one
would indicate multiple-copy involvement. Our result of log plots
dilution factor versus virions assembled support the
conclusion that the stoichiometry of pRNA in DNA packaging is between
five and six.
The stoichiometry of pRNA was also investigated by the
mixing together of inactive mutant pRNAs, each having interactive
complementary loops, in DNA packaging reactions to determine the common
multiples (24, 25). Since infectious virions could be produced by
mixing two inactive pRNAs with interlocking loops, we showed that the stoichiometry of the pRNA is a multiple of two (24, 25). Likewise, since infectious virions could also be produced by mixing together another set of three inactive pRNAs with interlocking loops, we showed
that the stoichiometry of the pRNA is also a multiple of three.
Therefore, we confirmed that the stoichiometry of pRNA in DNA packaging
is the common multiple of 2 and 3, that is, 6 or 12. Together with the
results from binomial distribution and serial dilution analyses (23,
71), we confirmed that the stoichiometry of pRNA was six.
The requirement of six pRNAs in phi29 DNA packaging is supported by the
finding that a pRNA dimer is the building block in the assembly of pRNA
hexamers (26). We found that the sequence in the assembly of hexamers
is dimer tetramer hexamer. The low resolution three-dimensional
structure of pRNA monomers and dimers has been shown by cryo-atomic
force microscopy (26, 27). Monomers exhibit a "check mark" shape,
while the dimer displays an elongated shape, with a size approximately
twice as long as the monomer (27).
The finding that phi29 RNA forms hexamers as part of an ATP-driven DNA
translocation machinery (24, 25, 28) has suggested commonalties between
viral DNA packaging and other universal DNA/RNA-tracking/riding processes, including DNA replication (29) and RNA transcription (24,
30). The DNA/RNA-tracking/riding enzymes include helicases (31-34),
enhancers (35), Escherichia coli transcription terminator Rho (36), yeast PCNA, and DNA polymerase III holoenzyme (37), each of
which also forms a hexameric complex or shape. Viral DNA packaging,
cellular DNA replication, and RNA transcription are all involved in the
relative movement of two components, one of which is nucleic acid. It
would be intriguing to show how the phi29 pRNA may play a role that is
similar to that of protein enzymes. It is speculated that
transportation of macromolecules by RNA complex, assembled via
intermolecular lop/loop interaction, exists in the life cycle of
eukaryotic cell differentiation (38).
We have determined the pathways and conditions for the assembly of
functional pRNA hexamers, where dimers serve as the building blocks
(26). In this study, the three-dimensional structure of the monomer and
the dimer was probed by chemical modification interference,
site-specific photoaffinity cross-linking, and cryo-AFM. This paper
provides a first report of pRNA three-dimensional structural constraints.
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EXPERIMENTAL PROCEDURES |
Synthesis and Purification of pRNAs--
pRNAs were prepared as
described before (39-41). Briefly, plasmid DNA was used as a template
for polymerase chain reaction (PCR) to prepare DNA templates for
in vitro transcription reaction. The primers used to produce
DNA template, as well as reverse transcriptase primer extension, are
listed in Table I. The PCR
products were purified using Qiaex II (Qiagen, Inc.) and made
ready for transcription with a T7 Ribomax transcription kit
(Promega, Inc.). Preparation of covalently linked dimer has been
described previously (26).
After synthesis, pRNAs were treated with RNase-free DNase I and then
subjected to 8 M urea, 8% polyacrylamide gel
electrophoresis. Bands of correct size visualized by UV shadow were
excised from the gel, and the RNA was eluted overnight at 37 °C in
0.5 M ammonium acetate, 0.1% SDS, 0.1 mM EDTA.
After elution, the pRNAs were ethanol-precipitated, washed with 70%
ethanol, and resuspended in nuclease-free H2O. Secondary
structure predictions for the pRNA were made using the method of Zuker
(42).
Chemical Modification Interference (72)--
Two pRNAs 5'/3'
B-a' and 23/97 A-b' (Fig. 1) were used to produce dimer (41). However,
only pRNA 5'/3' B-a' was modified by chemicals. In addition, primers
used in reverse transcriptase primer extension were specific to pRNA
5'/3' B-a' only. This strategy was to avoid ambiguity primer extension results.
DMS--
Purified pRNA (15 pmol) was incubated in buffer D (50 mM sodium cacodylate, pH 7.0, 10 mM
MgCl2, 100 mM NaCl) in a final volume of 50 µl. One µl of DMS (diluted 1:3 in 100% ethanol) was added to the
reaction. Unmodified control RNA was prepared by including 1 µl of
100% ethanol in the reaction instead of DMS. The reactions were
incubated for 3 min at 37 °C. Reactions were stopped by the addition
of 6.5 µl of DMS stop buffer (1.0 M Tris acetate, pH 7.5, 1.0 M 2-mercaptoethanol, 1.5 M sodium acetate,
0.1 mM EDTA) and then incubated on ice for 10 min (43).
Reaction volumes were brought up to 200 µl with DEPC-treated water
and extracted once with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and once with an equal volume of chloroform:isoamyl alcohol (24:1), followed by ethanol precipitation at  20 °C for several hours. Alternatively, the reactions were ethanol-precipitated directly after termination of the reaction. Pelleted RNA was
resuspended in 8 µl of DEPC-treated water.
CMCT--
Purified pRNA (15 pmol) in buffer C (50 mM
sodium borate, pH 8.0, 20 mM magnesium acetate, 100 mM NaCl) at a final volume of 25 µl was mixed with 25 µl of CMCT (12 mg/ml in buffer C). For unmodified control RNAs, 25 µl of buffer C was added instead of CMCT. Reactions were incubated
for 30 min at 37 °C and phenol-extracted and/or ethanol-precipitated
as described for DMS modification.
Isolation of Top and Bottom Band--
1.5 µl of DMS or 25 µl
of CMCT at a concentration of 37 mg/ml was used to modify pRNA 5'/3'
B-a'. The modified pRNA was subjected to electrophoresis in 8 M urea, 8% polyacrylamide gel in TBE (89 mM
Tris borate, 2 mM EDTA, pH 8.0) buffer (39, 41). The
band was excised using UV shadow and passively eluted overnight at 37 °C in the elution buffer, followed by ethanol precipitation. The
modified pRNA was washed with 70% ethanol, and the pellet was
resuspended in DEPC-treated water.
An equal molar ratio of the modified pRNA was mixed with pRNA 23/97
A-b' in TBM (89 mM Tris borate, 5 mM
MgCl2, pH 7.6) buffer. The mixture was then run on 8% TBM
polyacrylamide gel at 100 volts at 4 °C. Gel was stained with
ethidium bromide for visualization. Top and bottom bands were excised
and passively eluted at 4 °C in the elution buffer, precipitated by
ethanol, and resuspended in DEPC-treated water. The top and bottom
bands were dialyzed against TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for 1 h before being used in
primer extension.
Preparation of Photoagent-containing Circularly Permuted pRNAs
(cp-pRNAs)--
Guanosine 5'-phosphorothioate-containing cp-pRNAs were
prepared by in vitro transcription of DNA templates with T7
RNA polymerase in the presence of 40 mM Tris-HCl, pH 7.5, 12 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 1 mM ATP, 1 mM CTP, 1 mM UTP, 0.2 mM GTP, [ -32P]GTP,
8 mM guanosine 5'-phosphorothioate, at 37 °C for
4 h. Transcripts were purified by electrophoresis through 8%
polyacrylamide, 8 M urea gels, viewed by autoradiography,
and passively eluted into 10 mM Tris-HCl, pH 8, 0.3 M sodium acetate, 1 mM EDTA, and 0.1% SDS.
Transcripts were ethanol-precipitated and dried in vacuo. Transcripts containing the 5'-terminal phosphate of 5'-guanosine monophosphorothioate were coupled to an azidophenacyl group (44).
Cross-linking--
For cross-linking, the conjugated cp-pRNA was
incubated in TMS (50 mM Tris-HCl, pH 7.8, 10 mM
MgCl2, 100 mM NaCl) and then exposed to UV
light (Phillips, UVB 20W-TL01, 311 nm) for 10-20 min at 0 °C. Under
these conditions, no photoagent-independent cross-links were detected.
The efficiency of intramolecular cross-links (Table
II) was measured as a fraction of
the total input azido-pRNA using densitometric readings of individual
intramolecular cross-linked bands.
Separation of Cross-links by Sucrose Gradient
Sedimentation--
To separate intermolecular from intramolecular
(monomeric) cross-links, linear 5-20% sucrose gradients were prepared
in TB (50 mM Tris-HCl, pH 7.6, 89 mM boric
acid) buffer. Purified cross-linked species were loaded onto the top of
the gradient and spun at 50,000 rpm for 15 h at 4 °C in a SW55
rotor. As sedimentation markers, both pRNA dimers and monomers were run
on identical gradients. After sedimentation, fractions were collected
at 12 drops each and subjected to scintillation counting.
In Vitro Phi29 Virion Assembly Assay--
The purification of
procapsids (18, 45, 46), gp16, DNA-gp3 (47); the preparation of neck
and tail proteins (48, 49); and the assembly of infectious phi29 virion
in vitro (48-50) have been described previously.
Reverse Transcriptase Primer Extension--
RNA (1.5 pmol) was
mixed with 0.1 pmol of  -32P-end-labeled primer and
heated to 90 °C for 2 min. The mixtures were cooled to 30 °C in a
water bath (~1 h). RNA/primer mixtures were mixed with 0.5-1 unit of
avian myeloblastosis virus reverse transcriptase (Promega), 1 µl of
dNTPs (10 mM each), and 2 µl of 5× RT buffer (250 mM Tris-HCl, pH 7.9, 30 mM MgCl2,
10 mM spermidine, 50 mM NaCl) in a final volume
of 10 µl. Reactions were incubated at 55 °C for 30 min and stopped
by the addition of an equal volume of 2× loading buffer (98%
formamide, 10 mM EDTA, 0.01% bromphenol blue, 0.01%
xylene cyanol). Samples were heated to 90 °C for 2 min and placed on
ice before electrophoresis. Samples were subjected to sequencing-type
polyacrylamide gel electrophoresis, and dideoxy sequencing lanes were
run adjacent to experimental chemical modification reactions to
facilitate mapping of individual bases.
For cross-linked products, individual 5'-32P-labeled
oligonucleotide primers targeting various regions of the cp-pRNAs were hybridized to varying amounts of purified intramolecular cross-link species (75 °C, 2 min, then slowly cooled over 10 min to 37 °C). Oligonucleotides were extended by avian myeloblastosis virus reverse transcriptase at 45 °C for 20 min.
Cryo-AFM of pRNA Oligomers--
The procedure for cryo-AFM pRNA
image analysis has been reported previously (26, 27). The
oligomeric pRNAs were purified from native PAGE gel. Briefly, to
prepare the sample for cryo-AFM imaging, a piece of mica was freshly
cleaved and soaked with spermidine. Excess spermidine was removed by
repeated rinses with deionized water. A pRNA sample (10 µg/ml) was
applied to mica preincubated with TBM buffer. After 30 s, the
unbound pRNA was removed by rinsing with the same buffer. Before the
sample was transferred to cryo-AFM for imaging, it was quickly rinsed
with deionized water (<1 s), and the solution was removed with dry
nitrogen within seconds (51). All cryo-AFM images were collected at
80 K, as described elsewhere (52). Scan lines were removed by an
offline matching of the basal line. Calibration of the scanner was
performed with mica and 1-µm dot matrix.
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RESULTS |
Photoaffinity Cross-linking Strategy of pRNA Monomer Using
cp-pRNA--
Circular permutation allows the introduction of new 5'/3'
termini of pRNA while maintaining the correct folding of RNA molecule (40, 53, 54). Two tandem pRNA coding sequences separated by a
three-base sequence were cloned into a plasmid (40, 55). PCR primer
pairs complementary to various locations within the tandem pRNA coding
sequences were designed to synthesize PCR fragments for transcription
of cp-pRNA. We have shown that nonessential bases or their adjacent
bases can be used as new termini for constructing active cp-pRNA. The
circular permutation system greatly facilitates the construction of
mutant pRNA via PCR and the labeling of any specific internal base by
radioactive or photoaffinity agents (Fig.
1).
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Fig. 1.
Secondary structure of circularly permutated
pRNA and the locations of photoagent attachment sites indicated by
filled boxes (A). The numbering of pRNA is that
of the native sequence. Non-native nucleotides included in the
circularly permuted pRNA are the three A's underlined. The
procapsid binding domain and the DNA translocation domain are marked
with bold lines, and the four bases in the right
and left loops responsible for inter-RNA interactions are
boxed and in bold. Secondary structure of pRNA
5'/3' B-a' (B) and pRNA 23/97 (C), which interact
intermolecularly to form dimer are shown. The truncated pRNA 23/97 was
shown to be the smallest molecule that able to retain dimerization
(41).
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We performed photo-affinity cross-linking by attaching a photosensitive
agent to the 5'-end of the pRNA. The locations of the new end points in
cp-pRNAs used in this analysis were selected primarily due to their
ability to maintain wild-type activity as well as for their strategic
positions in the secondary structure (40, 55, 56). One of the sites is
located within the terminal helix necessary for DNA packaging, while
two of the other sites chosen are located within interior sequences
involved in procapsid binding. It was expected that data from these
constructs would provide structural information regarding the two
functional domains and their position relative to one another within
the pRNA. It has been reported that cp-pRNAs form the native structure,
while the 5' and 3'-ends are relocated (40). Nevertheless, it was important that the cp-pRNAs studied here reflect the native pRNA structure accurately. Previous analysis of the three cp-pRNAs chosen
for this study has revealed that these cp-pRNAs possess both wild-type
procapsid binding and DNA packaging activity (40, 55).
Cross-linking of Photoagent-modified cp-pRNA Monomers--
For
cross-linking, an azidophenacyl (APA) group was attached to the 5'-end
of individual cp-pRNAs. For the 5' modification, the APA group was
attached to cp-pRNAs containing a 5'-phosphorothioate incorporated
during transcription (44). Inclusion of guanosine monophosphorothioate
in transcription reactions results in its incorporation only at the
5'-end, because nucleoside monophosphates can initiate transcription,
but are unable to be utilized for elongation by T7 RNA polymerase. The
phosphorothioate sulfur provides a unique site on RNA for the
attachment of the azidophenacyl group. The photoaffinity-modified
cp-pRNAs are subsequently UV-irradiated, thus converting the azido
group to a highly reactive nitrene and able to insert into a variety of
covalent bonds. An APA derivative was used to provide long range
cross-links to identify proximal regions within or between structural motifs.
To monitor the cross-linking reactions, UV-treated
photoagent-modified cp-pRNAs were resolved on denaturing
polyacrylamide gels (PAGE). The cross-linked species appeared as bands
migrating more slowly than uncross-linked RNA (Fig.
2). As in previous analysis (56),
cross-linking occurred exclusively via the azidophenacyl moiety,
because unconjugated RNA did not form cross-links (data not shown). The
conversion rate of pRNAs containing the photoagent into cross-linked
species was 8-18% (Table II). These individual cross-linked species
are designated numerically according to their migration rate. For
example, the cross-linked species with the slowest and faster migration
rate using cp-pRNA 75/71 is denoted apa75-1 and apa75-2,
respectively.
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Fig. 2.
Identification of cross-linked species.
Radiolabeled 5'-APA cp-pRNAs were untreated () or treated (+) with
311 nm light for 15 min. Cross-linked species, and uncross-linked RNA
were resolved on a denaturing polyacrylamide gel. Arrowheads
and numbers to the right of each pair of lanes
indicate the major cross-link species.
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Intramolecular cross-linking of 5'APA cp-pRNA results in the formation
of lariats, which appear as bands migrating more slowly than
uncross-linked RNA in denaturing PAGE. However, it has also been shown
recently that pRNA in the presence of Mg2+ interacts
intermolecularly (24, 25). It was also shown that intermolecular
cross-linked pRNAs exhibit slower migration than uncross-linked RNA
when resolved on denaturing PAGE (56). Therefore, separation of
cross-linked species from noncross-linked RNA by PAGE is insufficient
to differentiate between intra- and intermolecular cross-links. To
distinguish between intra- and intermolecular cross-links, cross-link
species were isolated and subjected to sedimentation through a 5-20%
sucrose gradient not containing Mg2+. In such a gradient,
intramolecular cross-links will sediment like that of monomeric pRNA,
while intermolecular cross-links will sediment like that of multimeric
pRNA complexes. From the results shown in Fig.
3, it is clear that all cross-links
obtained in this study sedimented identically to the monomer control
and thus represents intramolecular cross-links.
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Fig. 3.
Sucrose gradient sedimentation analysis of
cross-linked species to determine whether the
cross-linking is intra- or intermolecular. Purified
cross-linked conjugates were loaded onto a 5-20% sucrose gradient and
sedimented in an ultracentrifuge. All cross-linked samples sedimented
identical to monomeric pRNA (monomer) indicating intramolecular
cross-linking, while dimeric pRNA complexes (dimer) centered at
fraction 8.
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Additionally, the formation of intramolecular cross-links was tested by
assessing the sensitivity of the reaction to dilution. A dilution of
photoagent-modified RNA before UV irradiation would cause a decrease in
intermolecular cross-linking rates, whereas intramolecular cross-links
should remain unaffected. Dilution up to 100-fold before UV irradiation
of cross-linking reactions containing 5'-APA cp-pRNAs did not affect
the efficiency of formation of the cross-linked species (data not
shown) and implied that the cross-linking is intramolecular rather than intermolecular.
Analysis of Intra-pRNA Cross-linking Sites--
The particular
nucleotides cross-linked to the modified termini of the cp-pRNAs were
determined by primer extension. Individual intramolecular cross-linked
species were gel-purified and used as templates for primer extension
with reverse transcriptase. Reverse transcriptase terminates one
nucleotide 3' to cross-link sites in the RNA template (57-60).
Examples of this analysis for the cross-linked species, derived from
apa75-2, apa78-2, and apa108-2 are shown in Fig.
4. Extension products from cross-linked
cp-pRNA were compared with that from noncross-linked cp-pRNA to
identify the individual cross-linked nucleotides. For intramolecular
cross-linked conjugates (apa75-1, apa78-1, and apa108-1), the
cross-link sites were apparently located near the 3'-end of the
cp-pRNA, within the oligonucleotide primer binding site, and so could
not be mapped by primer extension. Table II lists the sites of
cross-linking in the species analyzed in this study. The spatial
distribution of the related helix is illustrated in Fig.
5 and will be further discussed below.
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Fig. 4.
Primer-extension mapping of cross-link
sites. Analysis of intramolecular cross-links from cp-pRNA 75/71
(A), cp-pRNA 78/77 (B), and cp-pRNA 108/107
(C). Lanes marked with ddG, ddA,
ddT, ddC, and N denote lanes
containing G, A, T, and C sequencing reactions and a control primer
extension with noncross-linked RNA, respectively. Numbered
lanes contain the products of primer-extension reactions using the
cross-linked species indicated in Fig. 3. Arrows indicate
the positions of terminations specific to the cross-linked RNA
templates, while squares identify nonspecific
terminations.
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Fig. 5.
Overview of cross-linking
results. Structural constraints from this study
are indicated by lines to connecting photoaffinity
attachment sites (filled boxes) and cross-linked bases
(brackets). The numbering of pRNA is that of the native
sequence. Helices are marked and are numbered as they occur 5' to 3',
e.g. helix 3 is the third helix from the 5'-end. The loops
of individual helices are marked as left, right,
and head (H) loops. The four bases in the right
and left loops responsible for inter-RNA interactions are
boxed and in bold.
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Procapsid Binding and DNA Packaging Activity of Intramolecular
Cross-linked Monomers--
To test for the ability of cross-linked
cp-pRNA to bind procapsids, intramolecular cross-linked species were
gel-purified and incubated with purified procapsids in the presence of
Mg2+. Control reactions containing noncross-linked cp-pRNA
incubated with purified procapsids and Mg2+ were also
performed. The cp-pRNA-enriched procapsid complexes were sedimented
through sucrose gradients, and procapsid binding efficiencies were
determined (Table II). The gel-purified intramolecular cross-link
species retain substantial procapsid binding activity.
To analyze the DNA packaging activity, intramolecular cross-links were
gel-purified and utilized in in vitro phi29 assembly (48,
49). Under identical conditions to those used in assessing the DNA
packaging activity of uncross-linked cp-pRNAs, intramolecular cross-links showed reduced activity with respect to their
uncross-linked counterparts. Although intramolecular cross-links
exhibited relatively poor retention of DNA packaging activity, cp-pRNA
cross-links retained good procapsid binding efficiencies (Table
II).
Probing the Structure of Dimer by Chemical Modification
Interference--
Previous work has identified the intermolecular
interaction between the right loop of one RNA molecule and the left
loop of another RNA molecule (24, 25, 61). This intermolecular
interaction between the loops for the formation of a hexamer was
refereed as "hand-in-hand" interaction (41). In addition, pRNA
dimer has been shown to be the building block for the formation of the hexameric complex. However, the exact pathway of the hexamer formation is still a mystery even though a model has been proposed (26).
To facilitate description of mutant RNAs, we use upper and lowercase
letters to represent sequences of the interacting right and left loop
(Fig. 1). The same letters in upper and lowercases indicate
complementary sequences, while different letters indicate noncomplementary loops. For example, pRNA A-a' represents a pRNA with
complementary right loop A (5'-GGAC48) and left loop a'
(3'-CCUG82), while pRNA A-b' represents a pRNA with
unpaired right loop A and left loop b' (3'-UGCG82). Dimeric
pRNA was produced via intermolecular interaction of the engineered
mutant pRNA A-b' and B-a'. All pRNAs used were circularly permuted pRNA
75/71 (40, 55) with G75 and C71 as new 5' and
3' ends, respectively, except when indicated otherwise.
To avoid ambiguities in primer annealing during primer extension, we
employed two pRNAs, a regular 120-base, pRNA 5'/3' B-a', and a
truncated version of the 75-base, 23/97 A-b'. Previously we have shown
that 23/97 A-b' is the smallest molecule competent in forming dimer
(41). The 120-base 5'/3' B-a' was treated with either DMS or CMCT and
then mixed with the 75-base 23/97 A-b' for dimer formation. The
concentration of the chemical is titrated to ensure that on the average
only one base is modified for each pRNA. Monomer and dimer were
separated and purified from native PAGE. If the modified base is
involved in dimer formation, pRNA 5'/3' B-a' carrying this modified
base would not be able to form dimer with 23/97 A-b' and thus would
stay as the bottom band (monomer). The two bands representing either
monomer or dimer were isolated and subjected to primer extension to
identify the modified bases. Both primers used for reverse
transcriptase are specific to 5'/3' B-a' but are not able to bind 23/97
A-b' to avoid ambiguous results in primer extension.
Examples of autoradiograms of chemical modification interference by
both primer P11 and P103-82 are shown in Fig.
6, A-C. Reverse transcriptase
stops one nucleotide prior to the site of modification. Nonspecific
nicks in the RNA or nonspecific stops or pauses in primer extension
caused by strong pRNA secondary structure were identified by comparing
the primer extension on unmodified 5'/3' B-a' monomer as control.
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Fig. 6.
Chemical modification interference with DMS
and CMCT. A, DMS with primer P11; B, DMS
with primer P103-82; and C, CMCT with primer P11. Examples
of autoradiograms of sequencing type gels of primer extension using
-32P-end labeled primers are shown. Reverse
transcriptase stops one nucleotide prior to modified bases. The
M() and M(+) indicate primer extension was
performed on unmodified and modified 5'/3' B-a' pRNA monomer,
respectively. The top represents the monomer that escapes
the modification and thus produces band shift (dimer). The
bottom represents the monomer that got modified and thus
could not form dimer. The asterisks before the base indicate
the base is not involved in dimerization. The black arrow
indicates a strong modification, the gray square for
moderate, and the double-lined arrow for weak modification.
The ddA, ddG, ddC, or ddT
indicates that primer extension was performed on unmodified 5'/3' B-a'
pRNA in the presence of the indicated dideoxynucleoside triphosphate to
facilitate precise base mapping. Due to the difficulty in reading
extension products of increasing size, only a portion of the gels are
shown, thus no full-length products are presented in these figures.
Please note that DMS modifies only A and C, while CMCT modifies only U
and G. The bases modified unspecifically in both top and bottom are not
marked.
|
|
Bases C85 and C84 showed a very strong
involvement in dimer formation as seen by the intensity of the bands.
Bases A90, A89, C88,
U59, G55, and U54 show moderate
involvement in dimerization. Bases C71, A70,
U69, A68, A66, and C65
show weak involvement in dimerization.
In general, bases on the left-hand side of the left loops and bases
on the right-hand side of the right loop seem to hold the pRNAs
together to form the dimer, as summarized in Fig.
7, B and C. A
series of U74, U73, U72 at the
bifurcation bulge does not seem to be involved in dimer formation. We
believe this bulge provides the flexible hinge for the pRNA to
fold.
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|
Fig. 7.
Summary of modification interference on pRNA
5'/3' B-a' (C). The black arrow,
gray square, and double-lined arrow indicate a
strong, moderate, and weak modification, respectively. A and
B summarized the chemical modification of monomer and dimer
as a comparison (27).
|
|
Integration of Probing Data from Both Monomer and Dimer Concerning
pRNA Structure--
Data from the photoaffinity cross-link of monomer
revealed that base 75 was cross-linked to bases 26-30, and base 78 was
cross-linked to base 31. Data from chemical modification revealed that
bases 75-80 and bases 30-34 were protected from modification by
chemicals (27). Chemical modification interference revealed that bases 72-81 were not involved in dimer formation. Therefore, it is concluded that bases 75-81 and bases 31-35 are proximate. The 9-Å
cross-linking distance imposed by the photoaffinity agent allows the
nucleotides to cross-link to bases three to four bases away in the
sequence. Interaction between sequences 75-81 and 31-35 will produce
a pocket for RNA-RNA interaction in dimer formation. Chemical
modification interference revealed that bases 45-71 and 82-91 were
involved in dimer formation. Fig. 8,
A and C, illustrates the interaction that might
have taken place in pRNA dimer.
View larger version (58K):
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Fig. 8.
A, Sketch identifies the
intramolecular cross-linking sites (identified by a line
with double arrowheads) between base G75 to
stretch of
A26U27G28U29G30
and between G78 to U31. The flexibility of the
bifurcation bulge (U74U73U72) at
the three-way junction facilitates the flipping of the left loop. Also
shown in the sketch is an intermolecular cross-linking between base
G82 to bases
A41G40G39C49G62C63C64
(56). B, a bead-and-wire model to illustrate pRNA A/b'
monomer. C, a bead-and-wire model to illustrate pRNA dimer
A/b'-B/a', based on the data of chemical interference and the
cross-linking experiments.
|
|
Cryo-AFM Images of Covalently Linked Dimers--
We have
reported that the covalently linked dimeric pRNA is active in phi29 DNA
packaging (26). It is interesting to find out whether the overall
structure of the covalently linked dimer is similar to the native
pRNA dimer formed through hand-in-hand interaction.
We used the cryo-AFM to directly visualize purified pRNA monomers and
dimers. Previously, we report the pRNA monomer (27) (Fig.
9A) folded into a
" "-shaped structure, while dimers of A-b'/B-a' complex have an
elongated shape (Fig. 9, B and C). The overall
length of a monomer was found to be 16.7 ± 0.9 nm. The dimer had
a length of 30.2 ± 2.5 nm with a width of 11.6 ± 1.4 nm
(27). Since the dimer is elongated, it appears that head to head
contact was involved in dimer formation, resulting in a complex almost
twice as long as a monomer. It was also shown that nucleotides of the
head loop in dimers were protected from chemical attacks, strongly
supporting a head to head contact in dimer formation, in addition to
right and left loop interaction. Cryo-AFM images of the fused dimer
(Fig. 9, B and C-I) exhibit a similar structure
to the noncovalently linked pRNA dimer (Fig. 9C, panels II
and III). The dimensions of the covalently linked fused
dimer are comparable with the previously reported dimer (26, 27).
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|
Fig. 9.
Cryo-AFM to compare the covalently linked
(fused) dimers (B and C, panel I)
with noncovalently linked native dimer (C, panel
II and C, panel III) of
pRNA. pRNA monomers are also shown in A. The striking
similarity between the covalently fused pRNA dimer and the native pRNA
dimer is uncanny, albeit the covalently linked dimer is slightly
thinner at the center. The size of the dimer is about twice the size of
the monomer (compare A and C).
|
|
| |
DISCUSSION |
Intramolecular cross-links exhibited relatively poor retention of
DNA packaging activity; cp-pRNA cross-links retained good procapsid
binding efficiencies (Table II). Thus, poor DNA packaging activities
exhibited by the intramolecular cross-linked species probably is due to
the loss of flexibility after cross-link. It is expected that
conformation change of pRNA is needed during the DNA translocation
process (20).
Although the mechanism of phi29 pRNA dimer formation is similar to that
of the kissing loop of HIV, the type of loop-loop interaction of phi29
pRNA is in some respects different from pseudoknots (62, 63) and
kissing loops (64-68). Pseudoknots involve the intramolecular
interactions within one single molecule. Kissing loops involve the
interaction of two self-complementary loops to form a dimer (69, 70).
Since phi29 pRNA form closed hexameric rings, the intermolecular
interaction of pRNA must require that each RNA molecule contribute one
loop to pair with the alternate loop of the next pRNA, as shown in Fig.
8. The key feature in the "hand-in-hand model" is that multiple
RNAs interact via alternate interlocking loops to form a closed ring,
while interaction of kissing loops refers to the formation of dimers,
not rings.
Such hand-in-hand loop-loop interactions may also play an important
role in other systems as well. For example, RNA-RNA interaction via
alternating loops has also been reported for bicoid mRNA
in Drosophila embryos (38). We speculated that the mechanism
of bicoid mRNA interaction and translocation might be
similar to that of phi29 pRNA (41). It is possible, although not
proven, that a bicoid mRNA may also form multimeric
rings to ride, track, or rotate along Staufen protein during its
transportation. Indeed, there is evidence that bicoid
mRNA can form multimers (Fig. 3 of Ref. 38).
Hand-in-hand interaction is the mechanism in pRNA hexamer
formation (24, 41). The pRNA dimer reported here also involved hand-in-hand interaction. In dimer, each pRNA only held hands of
"one" additional pRNA. However, in hexamers, each pRNA held hands
of "two" additional pRNAs. It seems paradoxical concerning the hand
interaction in dimer and hexamer. How to interpret the conclusion that
dimer is the building block for pRNA hexamer? We found that the pRNA
has a strong tendency to form a circular ring by hand-in-hand contact
regardless of dimer, trimer, or
hexamer.2 Therefore, a
conformational shift is expected during the transition from dimer to
hexamer. We speculate that dimer formation is a prerequisite to
generate an appropriate three-dimensional interface for procapsid
binding. One of the hands of the dimer would release after binding to
the procapsid. The dimer with a released hand is similar to the open
(linear) dimer that has been demonstrated to be unstable in solution
but was still active in procapsid binding and DNA packaging (41). The
release hand will serve as a welcoming hand to recruit the incoming
dimer. Such conformation shift could be the intrinsic nature of such
intriguing RNA that could bear the moving task in DNA transportation.
| |
ACKNOWLEDGEMENTS |
We thank Stephen Hoeprich and Dan Shu for
their help in preparing this manuscript and Dr. David Williams for the
drawing of Fig. 8A.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants GM59944 (mainly) and GM60529 (to P. G.) and by National
Institutes of Health Grant RR07720 (to Z. S.).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.
§
Supported by a fellowship from the Universiti Sains Malaysia.
¶
These two authors contributed equally to this paper. Both of
them can be regarded as the first author of this article.
Current address: Western Fisheries Research Center, 6505 NE
65th St., Seattle, WA 98115.
**
To whom correspondence should be addressed: Purdue Cancer Research
Center, B-36 Hansen Life Science Research Bldg., Purdue University,
West Lafayette, IN 47907. Tel.: 765-494-7561; Fax: 765-496-1795;
E-mail: guo@vet.purdue.edu.
§§
Current address: Dept. of Molecular Genetics and Biochemistry,
University of Pittsburgh Medical School, Pittsburgh, PA 15261.
Published, JBC Papers in Press, May 22, 2001, DOI 10.1074/jbc.M100045200
2
D. Shu and P. Guo, unpublished results.
| |
ABBREVIATIONS |
The abbreviations used are:
pRNA, packaging RNA;
AFM, atomic force microscopy;
PCR, polymerase chain
reaction;
DMS, dimethyl sulfate;
DEPC, diethyl pyrocarbonate;
CMCT, (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide
metho-p-toluene sulfonate);
cp-pRNA, circularly
permuted pRNA;
APA, azidophenacyl;
PAGE, polyacrylamide gel
electrophoresis.
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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.
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ABSTRACT
INTRODUCTION