(Received for publication, October 23, 1996, and in revised form, March 19, 1997)
From the Department of Biosciences, Teikyo University, 1-1 Toyosatodai, Utsunomiya-shi, Tochigi 320, Japan
We have analyzed one of the functional domains of
Q replicase, an RNA-dependent RNA polymerase of RNA
coliphage Q
. Deletion mapping analysis of the carboxyl-terminal
region of the
-subunit protein revealed that the terminal 18 amino
acid residues (positions 571-588) are dispensable for the replicase
reaction. Subsequent deletions up to the Ala-565 residue reduced the
RNA polymerizing activity of the replicase in vivo but
increased it in vitro. The mutant replicases with enhanced
in vitro RNA polymerizing activity were found to have
relaxed template specificity for ribosomal RNAs and cellular RNAs as
well as Q
RNA. Deletions beyond the Ile-564 residue abolished both
the RNA polymerizing activity and the binding ability to midivariant
(MDV)-poly(+) RNA, a derivative of a natural template for Q
replicase, MDV-1 RNA. These results suggest that the carboxyl-terminal
part of the
-subunit participates in RNA recognition of Q
replicase.
With the development of modern gene technology, it became feasible to analyze the relationships between the structures and functions of gene products. In the case of RNA coliphages, the complete nucleotide sequences of representative phage genomes were determined (1-4), and analysis of the structure of each gene also became possible. In attempts to clarify the functional domain of the polymerases, several polymerase genes have been extensively characterized.
An RNA-dependent RNA polymerase (RNA replicase) of RNA
coliphage Q consists of four subunits. Three of them are
host-derived proteins (ribosomal protein S1, and protein elongation
factors, EF-Tu and EF-Ts) (5). Only the
-subunit, which is composed of 588 amino acid residues, is encoded by phage RNA. The central region
of the
-subunit is well conserved in RNA coliphages, and this region
is thought to contain common structural features for such as the
assembly of the subunits and catalysis for RNA synthesis (6). A
sequence motif, Gly-Asp-Asp, which has been identified in
RNA-dependent RNA polymerases of many viruses and is
considered to be the active site for polymerization (7, 8), is located in the central part of the
-sububit protein (9). Recently, this
motif sequence of the Q
and polio virus 3Dpol proteins
was found to take part in the binding of metal ions necessary for RNA
polymerization (10, 11).
The Q replicase can specifically transcribe in vitro the
Q
RNA as well as the RNA from closely related phages (5). S1 and
another host protein, HF-I (12), are required for the recognition of
only Q
viral plus-strand RNA as a template. In contrast, Q
replicase lacking S1 and HF-I transcribes poly(rC), MDV-1 RNA, and Q
minus-strand RNA, as does the holoenzyme of Q
replicase (5). RNA
replicases from phages MS2, GA, and SP share the host subunits of Q
replicase except for HF-I, but they have the different template
specificity. Therefore, it is reasonable to think that some factor(s)
determining the enzyme specificity must be present in the
-subunit.
The -subunit proteins of MS2 and GA replicases are approximately
10% shorter than those of Q
and SP replicases, with most of the
extra amino acids being located in the carboxyl-terminal region (4).
Furthermore, the carboxyl termini of the
-subunit are more divergent
in sequence (Fig. 1). It is therefore likely that this
region may contribute to template discrimination.
To clarify in detail the physiological role of the carboxyl-terminal
part of the -subunit in the Q
replicase reaction, we constructed
a series of plasmids harboring the
-subunit gene with deletions at
its 3
-terminal end and examined the effects of the deletions on the
enzyme functions. The carboxyl-terminal 18 amino acid residues were
found to be dispensable for the replicase reaction, but a larger
deletion made the enzyme defective.
Escherichia coli
A/ (sup pro/F+) and BE110
(sup+ rel-1 T2r
tonA22 phoA4 Hfr) were used as indicators. E. coli
594/F
lacIq (sup+
gal Strr
recA44/F
lacIq
lac+ proAB) was used as a recipient for
transformation and for growing phages. Phage Q
sus51 was described
previously (13). Plasmid pRQ1, which contains the wild-type
-subunit
gene of Q
replicase, was used to construct plasmids carrying various
sizes of the replicase gene lacking the 3
-terminal region. Plasmid
pUC-MDV-LR carrying the segment corresponding to MDV-poly(+) RNA was
described previously (10). MDV-poly(+) RNA is 244 nucleotides long and
is a derivative of naturally occurring MDV-1(+) RNA (14), for which
Q
replicase exhibits strong template activity. Plasmid ZpQ
-32
(15) was provided by C. Weissmann.
Poly(rC) and DNase I were purchased from
Pharmacia Biotech Inc. [-32P]ATP,
[
-32P]dCTP, [
-32P]GTP, and
[
-32P]UTP were obtained from Amersham Corp.
Restriction endonucleases were purchased from TaKaRa Shuzo. AmpliTaq
DNA polymerase was from Perkin-Elmer. The 16 and 23 S ribosomal RNAs of
E. coli MRE600 were from Boehringer Mannheim.
Plasmids carrying the -subunit gene, of which the
3
-terminal end was shortened to various extents, were constructed
using the polymerase chain reaction method. Oligonucleotide RQ1-f2 has a nucleotide sequence corresponding to positions 3328-3351 of the Q
cDNA sequence including the cleavage site for the restriction enzyme, XhoI, and was used as a forward primer (Table I).
The oligonucleotides listed in Table I were used as
reverse primers, containing an artificial stop signal leading to
premature termination of
-subunit protein synthesis and having the
site for BglII for inserting the DNA fragment into the
vector plasmid. Amplification of the DNA fragments was carried out
according to the manufacturer's instructions. The amplified DNA
fragments were digested with XhoI and BglII and
then ligated to plasmid pVAR-AD6 DNA (9), which harbors the
-subunit
gene lacking the 3
-terminal part and was previously cleaved with
XhoI and BamHI. The nucleotide sequences of the
amplified DNA regions were analyzed by the chain termination method
(16) after the DNA fragments had been subcloned into the M13 phage. The
plasmids constructed were designated according to the oligonucleotides
used as the reverse primers.
|
E. coli 594/F
lacIq cells carrying the plasmids were grown at
37 °C in YT broth (0.8% tryptone (Difco), 0.5% yeast extract, 0.5% sodium chloride) containing 10 mM CaCl2
to a cell density of 3 × 108 cells/ml, at which time
phage Q
sus51, which is defective in the
-subunit gene, was added
at a multiplicity of infection of 0.1. After 10 min at 37 °C, the
cells were spun down and resuspended in an equal volume of YT broth.
The cell suspension was then diluted 104-fold with YT broth
containing 2 mM
isopropyl-1-thio-
-D-galactopyranoside, and the
incubation was continued at 37 °C for 90 min, at which time
chloroform was added. An aliquot (100 µl), after appropriate dilution, was immediately mixed with a large excess of a stationary culture of E. coli A/
or BE110 and then plated onto a YT
broth-agar plate.
E. coli 594/F
lacIq cells carrying the plasmids were grown in
5 ml of YT broth containing 50 µg/ml ampicillin to a cell density of
2 × 108 cells/ml, at which time 2 mM
(final concentration) of
isopropyl-1-thio-
-D-galactopyranoside was added to the
medium, and the incubation was continued for another hour. Cell lysates
were prepared according to Ball and Kaesberg (17). Aliquots (5 µl) of
the lysates were analyzed by SDS-polyacrylamide gel electrophoresis to
demonstrate that a similar amount of
-subunit protein was present in
each lysate (Fig. 2). For the gel retardation assay, the
lysates were centrifuged at 30,000 × g for 30 min at
4 °C, and the supernatants were used.
Assay for Polymerase Activities Using Cell Lysates
The
wild-type and mutant polymerase activities were measured using
poly(rC), MDV-poly(+) RNA, or Q RNA as described previously (10),
with slight modifications. The standard reaction mixtures (50 µl)
each contained 125 mM Tris-HCl (pH 8.0), 25 mM
2-mercaptoethanol, 10 mM MgCl2, 50 mM phosphoenol pyruvate, 10 µg/ml pyruvate kinase, 74 units/ml DNase I, 10 µg/ml rifampicin, and 5 µl of cell lysate. For
the poly(rG) experiment, the mixture also included 0.4 mM GTP, 20 µg/ml poly(rC), and 740 kBq/ml [
-32P]GTP.
The MDV-poly RNA or Q
RNA reaction mixture contained 0.8 mM each ATP, CTP, and GTP, 0.1 mM UTP, 20 µg/ml MDV-poly(+) RNA or 68 µg/ml Q
RNA, and 1.48 MBq/ml
[
-32P]UTP. Incubation was performed at 35 °C for 15 min (MDV-poly RNA polymerase assay) or at 37 °C for 60 min (Q
RNA
polymerase assay). Aliquots (10 µl) of the mixtures were applied to
3MM Whatman filter paper discs. Incorporated radioactivity was
determined by liquid scintillation counting (10). In control reactions, 100 ng of purified Q
replicase was exogeneously added to the lysates
of host cells carrying pUC8. From the control assay results, it was
determined that 5 µl of the lysates of cells carrying pRQ1 contained
the equivalent of 271 ng (Q
RNA polymerase activity) or 247 ng
(MDV-poly RNA polymerase activity) of pure Q
replicase.
In this paper, MDV-poly(+) RNA represents an RNA that was transcribed from pUC-MDV-LR DNA by T7 RNA polymerase in vitro.
Northern Blot AnalysisE. coli 594/F
lacIq cells were grown and infected as described
under "In Vivo Complementation Analysis" except that the phage was
added at a multiplicity of infection of 3. After 10 min at 37 °C,
the cells were spun down and resuspended in an equal volume of YT broth
containing 2 mM
isopropyl-1-thio-
-D-galactopyranoside, and then the
incubation was continued for the indicated times. RNAs were extracted
with phenol and precipitated with ethanol in the presence of 0.8 M LiCl. The pellets were washed twice with 70% ethanol and
then dissolved in distilled water. A 5-µg sample of the extracted RNA
was denatured with glyoxal and dimethyl sulfoxide and then applied to a
1.0% agarose gel.
Electrophoresis, transfer to a nylon membrane (Biodyne A, Pall Ultrafine Corp.), and hybridization were performed as described previously (13).
A 1.2-kilobase pair XhoI-BamHI fragment of
ZpQ-32 DNA, which lies within the maturation (A2) gene, was labeled
using E. coli DNA polymerase I (Klenow fragment), random
oligonucleotide primers (nonamers), and [
-32P]dCTP and
then was used as the hybridization probe to detect Q
viral RNA.
The 5-end of MDV-poly(+) RNA was
labeled as described previously (10). Ice-cold binding mixture (15 µl) comprised 50 mM Tris-HCl (pH 7.5), 2 mM
MgCl2, 0.1 mM dithioerythritol, 5% glycerol, 1.5 × 10
2 pmol of 32P-end-labeled
MDV-poly(+) RNA, and 1 µl of various cell lysate supernatants. After
20 min of incubation at 5 °C, samples (10 µl) were applied to a
5% polyacrylamide gel at 4 °C as described by Werner (18). The gel
was then dried and subjected to autoradiography.
The wild-type and DS567
replicases were purified from E. coli 594/F
lacIq cells carrying the plasmids according to
Kajitani and Ishihama (19). For the Q
RNA polymerase assay, the
standard reaction mixtures (50 µl) each contained 80 mM
Tris-HCl (pH 7.5), 12 mM MgCl2, 12 mM 2-mercaptoethanol, 0.8 mM each ATP, CTP, and
GTP, 0.1 mM UTP, 74 kBq of [
-32P]UTP, and
200 ng of the replicase. Incubation was performed at 37 °C for 15 min. Incorporated radioactivity was determined as described under
"Assay for Polymerase Activities Using Cell Lysates."
We first
investigated whether or not the mutated -subunit proteins could
complement the activity of phage Q
sus51 replicase, an amber mutant
of the
-subunit gene. E. coli 594/F
lacIq cells carrying the mutant plasmids were
infected with phage Q
sus51, and then proliferation of the phage in
the cells was examined.
As shown in Fig. 3A, as long as the amino
acid deletion was less than 19 residues from the carboxyl-terminal end
of the -subunit protein, cells carrying each plasmid, pRQ1-DL585,
-DQ578, -DV576, -DA572, and -DV570, produced as many progeny phages as
did cells carrying the wild-type plasmid pRQ1 (Fig. 3A).
However, when the deletion extended beyond the Val-570 residue, progeny
production steadily decreased. Furthermore, when the deletion reached
the Ile-564 residue, the phage production in cells carrying larger deletion plasmids was abolished completely.
Northern Blot Analysis
To investigate the cause of the poor
phage production in cells carrying mutant plasmids (Fig.
3A), we extracted RNAs from phage Qsus51-infected cells,
and subjected the RNAs to Northern blot analysis.
As shown in Fig. 4, the 32P-labeled Q
cDNA probe strongly hybridized with Q
phage RNA (Fig. 4,
lane a) but did not hybridize with cellular RNAs at all
(Fig. 4, lane b), demonstrating that this cDNA probe
specifically detected Q
viral RNA. In the control cells carrying
pRQ1, viral RNA synthesis occurred at 20 min after the Q
sus51
infection, and many viral RNAs accumulated during another 20 min of
incubation (Fig. 4, lanes i and j). On the other hand, in the cells carrying plasmid pRQ1-DA565 or -DS567, in which phage production was greatly reduced (Fig. 3A), viral RNA
was found only a little even at 40 min after the infection (Fig. 4, lanes e-h). Since viral RNA synthesis did not occur in the
cells carrying pUC8 (Fig. 4, lanes c and d), the
residual viral RNA syntheses found in the cells carrying the mutant
plasmids were not due to the invading phage genomes.
Also seen were the hybridization signals at the positions near to those
of 16 and 23 S rRNAs (Fig. 4, lanes f and h-j).
These signals may represent the premature transcripts of Q RNA
because such small RNAs were observed in Q
RNA-dependent
RNA synthesis by wild-type or DS567 replicase (see Fig.
5C).
Q
Using lysates from cells
carrying various mutant plasmids described above, we examined Q
RNA-dependent RNA polymerizing activity.
As shown in Fig. 3B, as long as the amino acid deletion was
less than 18 residues, the polymerizing activity of lysates from the
cells carrying the mutant plasmid was similar to that of the control
cell lysate. However, when the deletion extended beyond the Val-570
residue of the -subunit, despite the fact that the cells expressing
the mutant replicases supported poor growth of the invading Q
sus51
phage (Fig. 3A), lysates of the cells carrying pRQ1-DR569,
-DS568, -DS567, or -DC566 synthesized much more RNA than did lysate
from cells harboring pRQ1. These results suggest that those deletions
did not affect the polymerizing activity of the replicase. When 25 or
more amino acids were deleted from the carboxyl terminus of the
-subunit protein, polymerizing activity was abolished. In addition,
lysates of cells carrying pUC8 exhibited little RNA polymerizing
activity.
To further analyze the characterization of the mutant replicase, we
purified DS567 replicase and compared the Q
RNA-dependent RNA synthesis by wild-type and DS567
replicases. As shown in Fig. 5A, when 16 ng of the template
Q
RNA was added to the reaction mixture, DS567 replicase synthesized
RNA in a similar manner and amount to wild-type replicase. However,
when 1 µg of the template RNA was incubated with DS567 replicase,
incorporation of [32P]UTPs was about 2-fold of that using
wild-type replicase. Fig. 5B shows the relationships between
Q
RNA-dependent synthesis and the molar ratio of Q
RNA to replicase (Q
RNA/replicase). Since Q
replicase binds to
Q
RNA at three sites (5), an excess amount of Q
RNA causes
binding of Q
replicase to two or three Q
RNA molecules, resulting
in inhibition of Q
RNA synthesis (20). Under our experimental
conditions, Q
RNA-dependent synthesis was found to be
maximum at a molar ratio of about 0.3 or 0.5 for the wild-type or DS567
replicase reaction, respectively. When the ratio was higher than 0.5, DS567 replicase synthesized RNA twice as much as wild-type replicase
did, which was consistent with the results in Fig. 4A. When
the 32P-labeled RNAs synthesized by wild-type or DS567
replicase at a molar ratio of 0.5 were compared by an agarose gel
electrophoresis, most of the 32P-labeled RNAs were found to
be similar in size to Q
viral RNA (Fig. 5C). These
results indicate that the polymerization activity of DS567 replicase
did not get damaged and suggest that DS567 replicase had an extra
ability to bind to Q
RNA.
It was puzzling
that lysates of cells carrying pRQ1-DC566, -DS567, -DS568, and -DR569
exhibited higher polymerizing activity in vitro with the
Q RNA template but that these cells were unable to support
effectively the proliferation of the invading phage, Q
sus51.
Taking account of the results in Fig. 5, the mutant replicases are
assumed to have more relaxed template specificity than the wild-type
replicase, so thereby they neglect Q RNA synthesis in cells infected
with phage Q
sus51. Therefore, we examined the polymerizing
activities of cell lysates using cellular RNAs as templates. As shown
in Table II, lysates of cells carrying pRQ1-DC566, -DS567, -DS568, or -DR569 incorporated more [32P]UTPs
than the control cell lysates carrying pRQ1 when ribosomal RNA or total
cellular RNA was used as template. In contrast, lysates of cells
carrying pRQ1-DA572 or -DV576, which exhibited lower Q
RNA
polymerase activity in vitro but supported progeny
production in Q
sus51-infected cells, had lower polymerization
activity than the control lysate in the cellular
RNA-dependent system.
|
To further determine the cause of the difference between the in
vivo and in vitro replicase reactions, we compared the
template specificity of purified wild-type and DS567 replicases. Q
replicase transcribes closely related phage RNAs but not other RNAs
such as ribosomal RNAs and unrelated phage RNAs (5). As shown in Table
III, when SP RNA, a closely related phage RNA to Q
RNA, was also used as a template, DS567 replicase incorporated more [32P]UTPs than the wild-type replicase, as in the case of
Q
RNA template. DS567 replicase also exhibited much higher RNA
polymerizing activity for 16 and 23 S ribosomal RNAs or total cellular
RNA as template, confirming the results of Table II. These results indicate that DS567 replicase relaxed specificity for not only Q
RNA
template but also cellular RNA templates.
|
Since Q
replicase lacking S1 and HF-I does not transcribe Q
RNA but can use
MDV-1 RNA and poly(rC) as template, we examined the effects of
deletions on MDV-poly RNA and poly(rG) polymerase activities. The
MDV-poly(+) RNA-dependent polymerizing activity of cell
lysates changed in a similar way to Q
RNA-dependent
polymerizing activity as the amino acid deletion extended into the
inner part of the
-subunit protein (Fig. 3B). When 25 residues were deleted, lysates of cells expressing mutant replicases
with larger deletions no longer showed the polymerizing activity.
The poly(rC)-dependent polymerizing activity of cell
lysates carrying pRQ1-DI564 decreased by 75% compared with the control cell lysates, as in the case of the MDV-poly(+) RNA template (Table IV). Furthermore, replacement of the carboxyl-terminal
Ile residue of DI564 replicase with Ala or a chemically similar Leu
residue reduced the poly(rG) and MDV-poly(+) RNA polymerizing
activities to background levels. These results indicate that the
sequence from Ile-564 to Ala-565 of the -subunit protein is
important for both the MDV-poly RNA and poly(rG) polymerase
activities.
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Q replicase binds to the
central region of MDV-1(+) RNA, in which nucleotides 81-126 are
identical with nucleotides 84-129 of Q
minus-strand RNA (21). To
determine whether or not mutant replicases without MDV-poly RNA
polymerizing activity can bind to a template, we examined a gel
retardation assay using 32P-end-labeled MDV-poly(+) RNA.
When the lysates of cells carrying pRQ1-DA565, pRQ1-DC566, or pRQ1 were
incubated with [32P]MDV-poly(+) RNA and then
electrophoresed on a polyacrylamide gel, the mobility of
[32P]MDV-poly(+) RNA was decreased (Fig.
6, lanes c and g-i). In contrast,
in the case of lysates of cells carrying pRQ1-DI564, which exhibited
poor polymerizing activity (Fig. 3B and Table IV), only a
small amount of [32P]MDV-poly(+) RNA migrated slowly
(Fig. 6, lane f), indicating that the binding of MDV-poly(+)
RNA to DI564 replicase was greatly impaired. Furthermore, in the case
of pRQ1-DQ562 or -DY563, a mobility shift was not observed anymore
(Fig. 6, lanes d and e).
Q replicase distinguishes its own Q
RNA and closely related
viral RNAs among natural RNAs. This discrimination involves the
recognition of a specific secondary or tertiary structure of the
template RNA. Q
RNA provides the replicase with three binding sites
(S-site, the initiation region of the coat gene; M-site, the internal
region of the
-subunit gene; and the 3
-terminal region of Q
RNA)
(3). However, little is known about the counterparts of the replicase
molecule responsible for the enzyme specificity, such as template
recognition or subunit assembly. In this paper, we propose that the
carboxyl-terminal part of the
-subunit of Q
replicase is one of
the candidates for functional domains determining the template
recognition from the following results.
DS567 replicase with deletions
of 21 amino acid residues at the carboxyl terminus had a relaxed
template specificity for a related phage RNA and cellular RNAs as well
as Q RNA (Tables II and III, Fig. 5). Furthermore, lysates of cells
expressing DR569, DS568, DS567, or DC566 replicase, which showed
in vitro higher polymerizing activity with the Q
RNA
template but failed to support progeny production in the cell,
exhibited higher polymerizing activity in cellular
RNA-dependent synthesis (Table II). In contrast, lysates of
cells expressing DA572 or DV576 replicase showed lower polymerizing
activity than the control lysate in Q
RNA or cellular RNA-dependent synthesis (Table II); however, these mutant
proteins complemented the activity of phage Q
sus51 replicase (Fig.
3A). These results suggest that in cells infected with phage
Q
sus51, a mutant replicase having a more relaxed template
specificity than that of wild-type replicase engaged in transcribing
cellular RNAs as well as Q
RNA and accordingly failed to perform the
task of polymerizing Q
RNA, indicating that the accessibility of
Q
replicase to cellular RNAs may be critical for phage growth.
DI564 replicase lacking
the carboxyl-terminal 24 amino acid residues of the -subunit protein
showed only a little activity in the MDV-poly RNA polymerase assay
(Fig. 3B). On removal of the terminal Ile residue from DI564
replicase, the resulting mutant, DY563, replicase lost the polymerizing
activity (Table IV). Furthermore, DI564 replicase showed reduced
binding ability to MDV-poly(+) RNA, and DY563 replicase abolished it
completely (Fig. 6). These results indicate that the failure of
MDV-poly(+) RNA-dependent RNA synthesis by DI564 replicase
was due to poor binding of the replicase to the RNA. Therefore, the
sequence from Ile-564 to Ala-565 of the
-subunit protein was
essential for Q
replicase to express MDV-poly RNA polymerase
activity, particularly for its binding to MDV-poly(+) RNA. The finding
that the insertion of a pentapeptide at the Ala-565 residue abolished
the MDV-1 RNA polymerizing activity of Q
replicase (6) agrees with
our results.
Recently, Brown and Gold (22) have proposed a three-site model of Q
replicase, according to which two RNA binding sites, site I on the S1
subunit and site II on EF-Tu, are responsible for RNA binding, and the
polymerase-active site on the
-subunit is for RNA synthesis. Site I
binds class I RNAs that possess single-stranded regions containing a
high fraction of A and C nucleotides such as Q
plus-strand RNA, and
site II binds class II RNAs that have polypyrimidine tracts such as
Q
minus-strand RNA or MDV-1 RNA. According to their model,
destruction of site II or removal of EF-Tu from Q
replicase will
result in the replicase without the MDV-1 RNA binding activity.
Therefore, the data in Fig. 6 that show the importance of the Ile-564
and Ala-565 residues of the
-subunit protein in MDV-poly(+) RNA
binding suggest that site II on EF-Tu interacts with these amino acid
residues to form an active Q
replicase molecule.
Our present results indicate that a change in the carboxyl-terminal
structure of the -subunit protein of Q
replicase could cause
relaxation of the template specificity and that this part of the
-subunit is responsible for recognizing MDV-poly(+) RNA. The
carboxyl-terminal region of the
-subunit protein is heterologous in
RNA coliphages. Phage RNA replicase is thus assumed to have evolved its
template specificity in part by altering the carboxyl-terminal structure of the
-subunit and simultaneously by keeping the
accessibility of the replicase to cellular RNAs at a low level.
We thank Akikazu Hirashima for reading the manuscript and for helpful discussions. We also thank our colleagues in our laboratory for help in isolating the mutant replicase clones.
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