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Journal of Virology, December 2001, p. 11373-11383, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11373-11383.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Autonomous Role of 3'-Terminal CCCA in Directing
Transcription of RNAs by Q
Replicase
David M.
Tretheway,1
Shigeo
Yoshinari,1 and
Theo W.
Dreher1,2,*
Department of
Microbiology1 and Center for Gene
Research and Biotechnology,2 Oregon State
University, Corvallis, Oregon 97331-3804
Received 15 June 2001/Accepted 23 August 2001
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ABSTRACT |
We have studied transcription in vitro by Q
replicase to deduce
the minimal features needed for efficient end-to-end copying of an RNA
template. Our studies have used templates ca. 30 nucleotides long that
are expected to be free of secondary structure, permitting unambiguous
analysis of the role of template sequence in directing transcription. A
3'-terminal CCCA (3'-CCCA) directs
transcriptional initiation to opposite the underlined C; the amount of
transcription is comparable between RNAs possessing upstream
(CCA)n tracts, A-rich sequences, or a highly
folded domain and is also comparable in single-round transcription
assays to transcription of two amplifiable RNAs. Predominant initiation
occurs within the 3'-CCCA initiation box when a wide variety of
sequences is present immediately upstream, but CCA or a closely similar
sequence in that position results in significant internal initiation.
Removal of the 3'-A from the 3'-CCCA results in 5- to 10-fold-lower
transcription, emphasizing the importance of the nontemplated addition
of 3'-A by Q replicase during termination. In considering whether
3'-CCCA could provide sufficient specificity for viral transcription, and consequently amplification, in vivo, we note that
tRNAHis is the only stable Escherichia coli
RNA with 3'-CCCA. In vitro-generated transcripts corresponding to
tRNAHis served as poor templates for Q replicase; this
was shown to be due to the inaccessibility of the partially base-paired
CCCA. These studies demonstrate that 3'-CCCA plays a major role in the control of transcription by Q replicase and that the abundant RNAs
present in the host cell should not be efficient templates.
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INTRODUCTION |
Genome replication among the
positive-strand RNA viruses is accomplished by sequential end-to-end
transcriptions, first of the encapsidated positive sense RNA and
subsequently of the newly synthesized negative-sense antigenome. Except
for viruses whose genomes are covalently linked at the 5' end to a
specialized protein, these transcriptions occur by de novo initiation
(9). For successful replication by this pathway,
transcriptional initiation must occur predominantly or exclusively at
the 3' ends of genome and antigenome RNAs, with minimal initiation
occurring internally or on other RNAs present within the cell. Q
replicase provides a convenient means to study the template properties
underlying these required specificities for a representative
positive-strand RNA virus.
Q replicase is the 4-subunit RNA-dependent RNA polymerase (RdRp)
enzyme complex that amplifies the 4.2 kb positive-strand genome of
bacteriophage Q, a phage infecting Escherichia coli (6, 34). Unlike the RdRp of any eukaryotic positive-strand RNA virus, Q replicase has been purified to homogeneity and
shown to be capable of supporting the full viral genome amplification cycle in vitro (6). This enzyme catalyzes de novo strand
initiation with GTP opposite a short cluster of C residues in the CCCA
3' termini that are a feature of both positive and negative strands of
almost all amplifiable templates described in the literature (24,
26, 40). After full-length transcription, termination is
accompanied by the addition of a nontemplated A residue
(6), thereby restoring the 3' A that was not copied into
the complementary strand.
While internal sequences whose removal decreases the transcription of
Q positive- and negative-sense genomic RNAs have been mapped
(27, 29), the precise features required for directing the
transcription of the genomic RNAs are unclear. Further, these elements
are not universally present in the wide variety of short RNAs
amplifiable by Q replicase (40). Indeed, it was
recognized several years ago (26) that the only feature
common to replicatable RNAs appears to be the CCCA 3' terminus; this
observation has held true with two exceptions, one being a variant Q
positive-sense RNA with a UCCA 3' end (28), the other
being a 6S RNA amplified by Q replicase with a GCCA terminus
(33). Nevertheless, it has not been demonstrated
experimentally whether the presence of a CCCA-terminal sequence is all
that is required for an RNA to be transcribed by Q replicase. Our
recent studies have shown that Q replicase can direct initiation
from every C2-4A repeat present in short linear
RNAs comprised of multiple C2-4A repeats (38), a property shared by the RdRps from turnip yellow
mosaic and turnip crinkle viruses (39). While these
studies suggested that a C2-4A element could act
as an independent initiation site, they did not resolve whether overall
transcription in these RNAs was supported by the reiterated C-rich
sequence motifs; Q replicase is well known for its ability to
transcribe poly(C) (6). Further, RNAs such as
(C2-4A)n are not
practical templates for amplification because of the large amounts of
internal initiation.
We set out in the present studies to investigate the ability of a
C2-4A sequence to act independently to direct
transcriptional initiation and to determine what sequences are
necessary to ensure that initiation occurs at the 3'-most C residue and
not at an internal site, thus ensuring the complete end-to-end copying
that is needed for sequence maintenance during replication. The results presented here provide a prescription for the minimal 3'-sequence requirement needed by Q replicase for efficient 3'-end initiation on
a template. This knowledge provides an explanation for the range of 3'
sequences observed for natural and model RNAs amplifiable by Q
replicase and insight into the mechanism that ensures the propagation
of full-length viral RNA with minimal interference in this process by
other RNAs present in the cell. Although there is evidence that
internal "promoter" elements specifically recognized by the
replicase function in positive-strand RNA viral systems (see, for
example, reference 12), including Q (the M-site on Q
positive-sense RNA [27]), such elements are not
absolutely required to ensure transcription by Q replicase. In this
system, we show that a small number of nucleotides at the 3' terminus of an RNA exert a crucial influence over the fate of that RNA as a template.
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MATERIALS AND METHODS |
Materials.
Q replicase, a generous gift from Michael
Farrell (Vysis, Inc.), was prepared by overexpression in E. coli (20). Synthetic DNA oligomers used for the
preparation of template RNAs were synthesized by Life Technologies,
Inc., or at the Central Services Laboratory of the Center for Gene
Research and Biotechnology, Oregon State University.
Preparation of RNA templates for Q replicase assays.
Template RNAs were prepared enzymatically with T7 RNA polymerase from
DNA templates comprising annealed DNA oligomers as described previously
(38) or from templates generated by PCR as described previously (30) in the case of E. coli
tRNAHis and its derivatives. All of the DNA
templates, other than those used to make CCCA9 and CCCCA7 RNAs (see
Fig. 3) and GGA(CCA)7X RNAs (see Fig. 5B),
contained modified 2'-O-methyl uridine and/or 2'-O-methyl guanosine at the two 5'-most nucleotide
positions to suppress the formation of n + 1 transcripts
(17). Transcripts were purified by 7 M urea-10%
polyacrylamide gel electrophoresis (PAGE) with single-base resolution.
Concentrations of RNA solutions were determined by spectrophotometry by
using extinction coefficients calculated by Oligo 6.0 software.
Q replicase assay and analysis of products.
Typical Q
replicase reactions (25 µl) contained 2.5 pmol (100 nM) of template
RNA and 1.25 pmol (50 nM) of Q replicase in 80 mM Tris-HCl (pH 7.5);
10 mM MgCl2; 1 mM dithiothreitol; 200 µM
concentrations each of ATP, GTP, and UTP; and 50 µM CTP, including 10 µCi of [
-32P]CTP. The experiments of Fig.
1 were conducted in the presence of 21 mM
MgCl2 as in previous studies (38);
we have adopted 10 mM MgCl2 as our standard
reaction condition to make our experiments more representative
of conditions used by other researchers. Incubation was for
10 min at 37°C, and products were recovered by phenol extraction and ethanol precipitation. At the time of ethanol
precipitation in the case of CCA9 RNA and its
GGA(CCA)7NNACCA derivatives, 250 pmol of DNA
oligomer complementary to the T7 transcript of CCA12 RNA was added;
this DNA promotes displacement of the template RNAs from the Q
replicase products during sample preparation for electrophoresis. In
other cases, 250 pmol of DNA exactly complementary to the RNA template
was added as described above, except for the RNAs analyzed in Fig. 6,
for which no antisense nucleic acids were used.
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FIG. 1.
Inactivation of the 3'-penultimate CCA initiation box
does not increase initiation from the 3'-CCA initiation box. RNA
variants derived from CCA9 RNA, bearing the mutations of the C residues
in initiation box #8 shown in panel A, were tested as templates with
Q replicase. RNAs were incubated in the presence of Q replicase
and [-32P]CTP for 10 min at 37°C as described in
Materials and Methods, except that MgCl2 was present at 21 mM. Products made from templates identified by their initiation box #8
sequence were separated by 12.5% denaturing PAGE (shown in panel B).
The numbers at the left identify the CCA repeat (initiation box
numbers) from which product strands originate, #9 representing the
3'-most CCA. Each CCA initiation box produces three products, marked a,
b, and c at the left, whose origins are explained in the text.
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Dried pellets recovered after ethanol precipitation were dissolved in
50 µl of 90% formamide-10 mM EDTA-0.02% dyes, boiled
for 5 min,
and ice-chilled, and then 10 µl was subjected to 7
M urea-PAGE.
After electrophoresis, gels were fixed and dried,
and radioactivity was
detected and analyzed with a PhosphorImager
with ImageQuant software
(Molecular
Dynamics).
Single-round transcription assays.
Assays were performed as
described above, except that CTP was initially withheld. After
incubation for 1 min at 37°C, polyethylene sulfonate (PES) was added
to 5 µg/ml, followed by 50 µM CTP, including 10 µCi of
[-32P]CTP. The assay was completed by
incubation at 37°C for 9 min, followed by product analysis as
described above.
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RESULTS |
A CCCA 3' terminus is superior to CCA at directing initiation
predominantly from the 3' end.
Our previous experiments have shown
that (CCA)n RNAs of ca. 30 nucleotides (nt) or
longer are good templates for Q replicase (38),
supporting a cumulative level of RNA strand initiation similar to that
observed with an amplifiable positive control RNA, DN3
(39). However, most initiation on such RNAs occurs
upstream of the 3' end (38) (see also Fig. 1, lanes marked CCA9), resulting in sequence loss that is incompatible with the requirements of viral RNA replication. More precisely, predominant initiation occurs opposite the downstream C of the 3'-penultimate CCA
repeat (CCACCA, in box #8; 59% of the initiation products were longer than 12 nt), with weaker initiation opposite the 3'-most C
(CCACCA, in box #9; 17% of initiations; Fig. 1, lanes 2, 8, 14, and 21). Note that the band heterogeneity present as bands 8a,
b, and c in the above lanes is principally due to differences at the 3'
end of the product, a finding we have reported previously (38) and reconfirmed in this study (not shown). Whereas
the longer CCA12 RNA (38) produces mainly doublets, CCA9
RNA yields the triplets labeled a, b, and c. Our studies indicate that
(i) the "a" band represents transcription to the 5' end of the
template, followed by addition of a nontemplated A (25,
36) (CCA 3' end), (ii) the "b" band represents a mixture of
full-length transcripts that fail to acquire an additional A (CC 3'
end) and those that terminate 1 nt early and acquire a nontemplated A
(CA 3' end), while (iii) the "c" band represents termination 1 nt
early with no adenylation (C 3' end). We have shown that this pattern
of products holds true with initiation from 3'-CCCA by
labeling RNAs AAA71, AAA51, and AAA31 (see Fig. 4)
with [-32P]CTP and inspecting RNase
T1-released fragments (results not shown).
To test whether the predominant initiation at CCA box #8 in CCA9 RNA
results from the suppression of initiation from the 3'-terminal
CCA box
#9, we studied the spectrum of transcription products
in a family of 15 templates in which the C residues of box #8
were mutated with single or
double substitutions to NNA. As shown
in Fig.
1, all single or double
mutations in CCA9 RNA resulted
in the almost complete loss of
initiation from box #8, with little
or no other changes in the spectrum
of initiations from other
sites. Most significantly, mutation of box #8
did not result in
increased and preferential initiation from the 3' end
(box #9).
The observations were uniform, regardless of whether the
substitution
of C was with the pyrimidine U or the purines A or G, at
position
5, position
6, or both. These results indicate that the
absence
of predominant initiation from the 3' end in CCA9 RNA is mostly
due to the absence of an appropriately strong initiation signal
and not
due to competition by an upstream
CCA.
Thus, although the sequence CCA clearly does constitute a functional
initiation box, the results of Fig.
1 indicate it is
relatively weak in
directing initiation from the 3' terminus of
an RNA. Indeed, RNAs
reported in the literature as being amplifiable
by Q
replicase
typically terminate in NCCCA (
26,
34,
40),
suggesting that
a 3'-CCCA rather than 3'-CCA is necessary to ensure
preferential
initiation from the 3' terminus. To test this idea,
we studied
transcription from a second family of templates based
on CCA9 RNA, in
which the CCACCA terminus was replaced with NNNCCCA.
Of the 14 3'-heptanucleotide sequences tested, 11 were derived
from the published
sequences of amplifiable RNAs (Fig.
2A).
As
is evident from Fig.
2B, a CCCA terminus ensures predominant
initiation
from the 3' end in the presence of a variety of
trinucleotide
sequences immediately upstream (mutations in
initiation box #8).
A total of 59 to 87% of all products longer than
12 nt initiated
from within the CCCA initiation box for all templates
tested,
except for UGGCCCA RNA (Fig.
2B, lane 12), which was a poor
template
overall (accessibility probing with RNases suggested that this
RNA exists in solution with some undetermined secondary structure,
which presumably interferes with template activity). In contrast,
59%
of the products longer than 12 nt transcribed from CCA9 RNA
originated
from the 3'-penultimate CCA initiation box (#8), with
only 20% of the
initiations occurring from the 3'-CCA (box #9).
No change in product
spectrum was observed when representative
RNAs from Fig.
1 and
2 were
analyzed under single-round transcription
conditions in the presence of
5 µg of PES/ml (data not shown).
There is therefore no indication of
preferential reinitiation
at certain sites.
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FIG. 2.
A CCCA 3'-initiation box directs strong 3'-terminal
initiation from a wide range of adjacent sequences. (A) RNAs in which
the 3'-CCACCA of CCA9 RNA was changed to 3'-NNNCCCA were incubated in
the presence of Q replicase and [-32P]CTP for 10 min at 37°C as described in Materials and Methods, with
MgCl2 present at 10 mM. As indicated, many of the
3'-heptanucleotide sequences were derived from the 3' termini of RNAs
exponentially amplifiable by Q replicase: Q genome(+), WSI
(26); Q genome() (34); MDV-1
(24); DN3 (40); RQ120 (21);
50#1, 50#2, and 77#1 (7); MNV-1 (2); SV7
(GenBank accession no. L07339); and SV11 (GenBank accession no.
L07337). (B) Analysis of transcription products labeled with
[-32P]CTP and separated by 12.5% denaturing PAGE,
with templates identified by the sequence of their modified initiation
box #8. The relative levels of transcription (with reference to box #8
of CCA9) originating from box #9 of each RNA is given at the foot
of each lane (average of three experiments; typical standard
deviation = 10 to 20%). Below the panel is shown the proportion
(% of total) of transcripts >12 nt in length originating from box #9
(box #8 for CCA9).  , Quantitation of transcription from box #8 in
the case of CCA9 RNA.
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Similar levels of initiation were observed when the CCCA 3'-terminal
initiation box was adjacent to various sequences (Fig.
2, mutated box
#8), indicating that these adjacent sequences have
little influence on
transcription by Q
replicase. The level of
initiation from CCCA was
similar to that from CCA box #8 of CCA9
RNA.
Relative initiation strength from 3'-most and 3'-penultimate
initiation boxes is influenced by the number of cytosine residues.
The results presented above show that a CCCA initiation sequence is
superior to CCA at the 3' end, although CCA provides strong initiation
from a position slightly internal to the 3' end. To explore the
relationship between initiation from these two positions, we analyzed
transcription from RNAs with different numbers of cytosines in either
the 3' or 3'-penultimate initiation boxes. The addition of increasing
numbers of C residues to box #9 of CCA9 RNA dramatically switches the
initiation preference from box #8 to the 3' end (Fig.
3B, lanes 1 to 5). Thus, the ratio of
products initiating from box #9 relative to box #8 increases from
0.29:1 for CCA9 to 1.1, 4.7, and 14:1 when the 3' box (#9) has the
sequences C3A, C4A, and C5A, respectively (Fig. 3B).
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FIG. 3.
C3-5A sequences provide optimal 3'
initiation sites. The RNAs shown in panel A were analyzed as
transcriptional templates for Q replicase (panel B) as described in
Fig. 2. The relative levels of transcription (% of total, with
reference to box #8 of CCA9) originating from boxes #8 and #9 of each
RNA are given below each lane (average of three experiments; typical
standard deviation = 10 to 20%); note that for lanes 14 and 15, the numbers refer to initiation from the 3' and 3'-penultimate boxes,
respectively. Below these figures are shown the proportions of
transcripts >12 nt in length originating from box #9 (as well as box
#8 for CCA9) as a percentage.
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In the absence of a functional 3'-penultimate initiation box (Fig.
3B,
lanes 8 to 11), predominant end-to-end transcription
is driven by 3'
initiation boxes with three, four, or five C residues.
In these cases,
80 to 90% of products longer than 12 nt originate
from the 3' end
(Fig.
3B). The level of transcription was some
30 to 60% higher from
3'-C4A or 3'-C5A than from 3'-C3A, indicating
that C strings longer
than three can provide modest additional
initiation strength.
Initiation from 3'-CCA is at about the same
low level in the presence
or absence of CCA at box #8 (Fig.
3B,
lanes 7 versus 8), indicating
that initiation boxes #8 and #9
act largely independently rather than
in competition (compare
also transcription from box #9 of Fig.
3, lanes
3 to 5 versus
lanes 9 to 11). The decreasing level of
transcription from box
#8 in lanes 2 to 5 of Fig.
3B is attributable to
the increasing
distance from the 3' end as C residues are added to box
#9. Thus,
box #8 of CCAC5A RNA (lane 5) is actually at the same
position
relative to the 3' end as box #7 of CCA9 RNA (lane 2), and
both
show similar amounts of
initiation.
Increased numbers of C residues at the 3'-penultimate initiation site
leads to a higher ratio of initiation from the 3'-penultimate
relative
to 3' boxes (Fig.
3B, lanes 16 versus 13). With more
C residues at both
sites, 3' initiation becomes relatively more
favored, although internal
initiation is still important (Fig.
3B, lanes 14 and 15). These results
indicate that the proportion
of end-to-end transcription is strongly
influenced by the C
nA
sequences present at and
adjacent to the 3' end. At both positions,
but particularly at the 3'
end, initiation is favored by a larger
number of C residues. Our
results refine previous findings (
15)
that somewhere
between 5 and 20 3'-C residues are able to activate
transcription of an
RNA by Q
replicase.
To what extent do the upstream CCA boxes of AAACCCA and related
RNAs contribute to transcription?
The template activities of
AAACCCA and related RNAs analyzed in Fig. 2 indicate the capacity of
3'-CCCA to independently direct initiation, unsupported by a
neighboring CCA box. However, we were interested to know whether the
seven contiguous upstream CCA boxes were important contributors to the
strong initiation from the 3' end. Such a contribution seemed
plausible in view of the well-known ability of Q replicase to
transcribe poly(C) but not other homopolymers (6) and the
prevalence of pyrimidine-rich sequences in replicons (7).
To experimentally address this question, we analyzed transcription from
variants of AAACCCA RNA in which (CCA)3 segments were
changed to A4UA4 (RNAs AAA31 to AAA35),
(CCA)5 segments were changed to
A4UA5UA4 (RNAs AAA51 to AAA53), and (CCA)7 to
(A2UA2UA)3 (AAA71 RNA) (Fig.
4A). The interspersed U residues were
designed to prevent the replicase slippage anticipated on longer A
tracts. As well as testing a role for C clusters as nonspecific
transcriptional enhancers, these RNAs permitted testing a role for a
short C cluster at an appropriate spacing upstream of the 3'-initiation
box in enhancing transcription (proposed in reference
18), perhaps by binding to so-called Site II of the
EF-Tu subunit (8).
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FIG. 4.
Upstream CCA boxes contribute little to initiation from
a 3'-CCCA sequence. (A) Derivatives of AAACCCA RNA in which CCA boxes
#1 to #7 have been replaced progressively with A-rich sequences
(underlined). (B) Analysis of the same RNAs as transcriptional
templates for Q replicase, performed as described in Fig. 2. The
dots placed to the left of lanes indicate absent signals from the
mutated initiation boxes. The relative level of transcription
(percentage of total, with reference to box #8 of CCA9) originating
from box #9 of each RNA is given below each lane (average of three
experiments; typical standard deviation = 10 to 20%), as is the
proportion (percentage of total) of transcripts >12 nt in length
originating from box #9.
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All of the modified RNAs showed robust predominant initiation from the
3' end (Fig.
4). Similar results were obtained with
variants in which
CCA boxes #1 to 7 were individually changed
to AAA (not shown). Most
convincingly, AAA71 RNA (Fig.
4B, lane
12), which lacks any C residues
upstream of the 3'-CCCA initiation
box, supported similar levels of
end-to-end transcription as the
AAACCCA parental RNA (lane 3);
essentially all transcription was
end to end. Similarly strong
end-to-end transcription was observed
from AAA72 RNA (Fig.
5, lane 3; 170% transcription relative
to
box #8 of CCA9), an RNA related to AAA71 RNA, but with only
three
U residues in the A-rich tract upstream of the 3'-CCCA. Mutation
of each CCA box individually from the various RNAs tested in Fig.
4
resulted in the loss of initiation from that site (indicated
with a dot
next to each lane) but only modest (if any) changes
in the initiation
levels from adjacent CCA boxes. These results
indicate that all CCA
boxes serve as independent initiation sites
and that the upstream CCA
tract provides little or no enhancement
of transcription initiation
from the 3'-CCCA. The notion of a
role for an upstream C cluster
(
18) has been questioned by the
inapparent conservation of
such a feature among RNAs amplifiable
by Q
replicase
(
26).
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FIG. 5.
Role of the 3'-terminal A residue. Two families of RNAs
are analyzed as transcriptional templates for Q replicase as
described in Fig. 2. In lanes 2 to 4, derivatives of AAA72 RNA with the
indicated 3' ends are analyzed: the AAA72 RNA family has the sequence
GGA5UA6UA5UA6 upstream
of the indicated C-rich initiation box. In lanes 6 to 9, derivatives of
GGA(CCA)7X RNA, with the indicated 3' ends representing X,
are analyzed. The major end-to-end transcription product is marked N at
the right side of the figure, with the upper band evident in lanes 8 and 9 marked N+1. The entire cluster of bands originating from the
initiation boxes of the RNAs in lanes 6 to 9 is bracketed and labeled
"C." The relative percent transcription yields are given below each
lane, separately for C, N, and N+1 in panel B. The yields of products
in lanes 3 and 6 were 170 and 125%, respectively, relative to
transcription from box #8 of CCA9 RNA. The RNAs in lanes 6 to 9 were
previously analyzed (38) at 21 mM MgCl2, which
favors internal over 3'-end initiation.
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Contribution of the 3'-A to initiation box function.
The role
of the 3'-A in the CCCA 3'-initiation box was tested by observing
transcription from AAA72 RNA with the 3'-A present or absent
(3'-CCCA and 3'-CCC termini, respectively). AAA72 RNA lacking the
3'-A yielded less than one-tenth the amount of transcript produced from
full-length AAA72 RNA (Fig. 5, lanes 3 and 4), indicating the
importance of the addition of nontemplated A by Q replicase to the
3' end of product strands. Transcription from the 3'-CCC terminus was even poorer than from AAA72 RNA terminating in 3'-CCA (Fig. 5, lane 2). Comparison of the same three termini on two other
RNAs likewise emphasized the importance of the 3'-terminal A residue:
with both GGA(CCA)7AAAX and
GGA(CCA)7CCUX RNAs, where X is CCA, CCCA,
or CCC, about one-fifth the level of transcription observed from CCCA
ends was observed for both CCA and CCC ends (not shown). Our results
are consistent with studies reporting 33% template activity for Q
genomic RNA lacking the 3'-A of Q genomic RNA (25),
suggesting fundamentally similar recognition of the 3' termini of the
viral RNA and the synthetic templates we have used here.
A specific role for A at the 3' end was tested with a
GGA(CCA)
7C
5N RNA
family, in which N was A, G, U, or C. Transcription
was strongest with
a 3'-A (Fig.
5, lane 6), and 56 to 66% that
level with the other
nucleotides at the 3' end (lanes 7 to 9)
when quantitated on the basis
of the entire cluster of bands derived
from the C-rich initiation box
(bracketed "C" in Fig.
5B). The
yield of full-length transcript
(band N) was only 34 to 35% from
RNAs with a 3'-C or -U (relative to
RNA with 3'-A), reflecting
the presence of considerable band
heterogeneity in the "C" clusters
of lanes 8 and 9. This
heterogeneity includes an additional band
N + 1, which could reflect
initiation opposite the additional
3'-pyrimidine, stuttering during
initiation, or a gel artifact:
despite the use of antisense DNA to
hybridize the template molecules
(see Materials and Methods), some
replicase products are harder
than others to completely denature, and a
shadow or faint band
at the N + 1 position is occasionally seen. For
the RNAs with
3'-U or -C (lanes 8 and 9), the band heterogeneity is
also expressed
as the N band being less prominent than the bands
immediately
below: we do not know whether this reflects staggered
initiation
at alternative C residues or a differential heterogeneity
introduced
during
termination.
In summary, a 3'-A is an important feature of a template with three C
residues in the 3'-initiation box, although it is less
important when
more C residues (five or six) are present. In the
latter case, a 3'-A
additionally has a potential role in minimizing
initiation
heterogeneity.
A 3'-CCCA initiation box alone supports transcription at levels
similar to that seen with replicon RNAs.
Having concluded from the
above study that no specific sequence other than a 3'-CCCA (or closely
related sequences) is required for transcription by Q replicase, we
wanted to test whether the transcription levels seen with our short,
linear RNAs are comparable to those supported by RNAs capable of
amplification. Transcription of CCA9, AAACCCA, and AAA71 RNAs (Fig. 4)
was compared with product synthesis from two replicon RNAs capable of
amplification by Q replicase: MDV and DN3 RNAs. Single-round
transcription was studied through the addition of the polymerase
scavenger PES (see Materials and Methods). In the presence of PES,
AAACCCA and AAA71 RNAs (each 31 nt long) yielded similar amounts of
transcript as DN3 RNA, a replicon of similar length (34 nt
[40]) (Table 1). Some 3.5 times more transcript was obtained from MDV RNA, a 225-nt-long replicon
(Table 1). We have previously observed a marked length effect with
short transcripts, CCA12 (39 nt) supporting about three times the
transcription derived from CCA9 RNA (38). The increased
transcription from MDV relative to AAA71 RNA may be due to its greater
length or other properties, such as the presence of a replicase
recognition site (22). Nevertheless, the model RNA AAA71
with transcription driven solely by a 3'-CCCA initiation box supports
transcription levels comparable to those of replicon RNAs. Similar
results were observed at 10-fold-lower template and replicase
concentrations of 10 and 5 nM, respectively (data not shown).
Can a 3'-CCCA transcription signal provide sufficient template
specificity in vivo?: the case of tRNAHis.
While the
results presented above indicate that an accessible CCCA positioned at
the 3' end of an RNA is sufficient to make that RNA a strong template
for Q replicase, we were concerned that this may not be sufficiently
specific to prevent transcription of some of the abundant stable RNAs
present in an E. coli cell, especially some of the tRNAs,
all of which end in the conserved 3'-CCA. Perusal of the sequences of
all E. coli tRNAs (32;
http://www.uni-bayreuth.de/departments/biochemie/trna/) and other RNAs
(Blattner et al., http://www.genome.wisc.edu/k12.htm) revealed that
only tRNAHis has a 3'-CCCA. Note,
however, that tRNAHis is also unique in
possessing an additional 5'-G relative to other tRNAs, resulting in the
underlined "discriminator base" being base paired (Fig.
6A).
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 6.
E. coli tRNAHis is a poor
template for Q replicase because its 3' terminus is unavailable. (A)
Sequence of tRNAHis (lacking posttranscriptional
modifications). The arrow marks the additional 5'-nucleotide (G) that
is unique to tRNAHis and that is absent from the RNA
template used in lane 3 of panel B. The bracket encompasses the 5'-GGU
missing from the template tested in lane 4. The additional 3' sequence
present on the RNA tested in lane 5 is shown in italics. (B)
Transcription products generated from the indicated templates after
incubation with Q replicase as described in Fig. 2, except that
analysis is by 10% denaturing PAGE. The relative molar transcription
levels (Rel. tr. [percent], with reference to box #8 of CCA9, 100*)
originating from each RNA is given at the foot of each lane (average of
three experiments; typical standard deviation = 10 to 20%).
|
|
To see whether tRNA
His could serve as a template
for Q
replicase, we synthesized by in vitro transcription a form of
the RNA
lacking the usual base modifications (Fig.
6A). Only weak
end-to-end
transcription was supported by the
tRNA
His transcript (4.5% yield relative to
transcription from box #8
of CCA9; Fig.
6B, lanes 1 and 2). To test
whether this weak transcription
was due to the poor accessibility of
the 3'-CCCA, transcription
was also tested from three variants which
either lack the 5'-G
or 5'-GGU or have an additional AACCCA at the 3'
end. Removal
of bases from the 5' end progressively resulted in
increased transcription
(Fig.
6B, lanes 3 and 4), while addition of the
3'-AACCCA resulted
in a still-higher level of transcription (78%
relative to box
#8 of CCA9). This latter level of transcription is
comparable
to that observed from AAA71 and other RNAs in Fig.
4 that
terminate
in 3'-CCCA, indicating that the body of the tRNA neither
enhances
nor represses transcription directed by the CCCA initiation
box.
These results demonstrate that host tRNA
His
is only a poor template for Q
replicase and that this is due
to the
inaccessibility of the initiation site due to base
pairing.
Transcription initiation from a 3'-UCCA terminus.
The results
shown in Fig. 1 to 6 clearly demonstrate the ability of 3'-CCCA and
related sequences with additional C residues to direct transcription by
Q replicase, explaining the presence of such sequences at the 3'
ends of almost all replicons. However, we know of two instances in the
literature that deviate from this rule: a host factor-independent
variant of Q RNA that is able to amplify in vivo and has a CCUUCCA
3' end (28) and a 6S RNA amplifiable in vitro with a GCCA
3' end (33). We have tested the ability of the UCCA
terminus to support transcription initiation by Q replicase with
GGA(CCA)7CCUUCCA RNA as a template.
This RNA supported a 98% end-to-end transcription level relative to transcription from box #8 of CCA9 RNA (Fig.
7), showing that at least with certain
adjacent sequences a UCCA 3' end is capable of supporting robust
initiation. The levels of initiation from internal CCA boxes was
elevated about twofold for this RNA relative to CCA9 RNA, suggesting
that the absence of competing initiation sites may be more important
with a UCCA 3' end in a replicon RNA.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 7.
Transcription from a 3'-UCCA terminus. Transcription
from GGA(CCA)7CCUUCCA and CCA9 RNAs by Q replicase was
compared as described in Fig. 2. CCA9 RNA (30 nt) is the shorter of the
two RNAs by 1 nt.
|
|
| |
DISCUSSION |
An accessible CCCAOH initiation box is sufficient to
promote robust transcriptional initiation by Q replicase.
The
results presented in Fig. 2 to 5, using short unstructured model
templates, show that a 3'-CCCA terminus can direct strong end-to-end
transcription by Q replicase in association with a number of
adjacent and upstream sequences. We have also demonstrated that a
3'-CCCA initiation box can function as the sole transcriptional control
element in a template. Related 3' termini with four or five C residues
direct somewhat more initiation (Fig. 3). A 3'-ACCA terminus is
relatively ineffective at directing initiation from the 3' end (Fig.
3), although UCCA can support significant initiation (Fig. 7). The
level of transcription observed from 3'-CCCA is approximately the same
regardless of whether the upstream region of the template is comprised
of CCA repeats, an A-rich sequence, or a mixture of the two (Fig. 4).
Similar levels of transcription were also observed when a structured
RNA (tRNAHis) that is itself a weak template was
provided with an accessible 3'-CCCA initiation site (Fig. 6). The
levels of transcription observed from the short linear RNAs with a
3'-CCCA initiation box are similar to those supported by RNAs capable
of amplification by Q replicase (tested in single-round
transcription assays; Table 1). These experiments provide a clear
demonstration of the importance of a 3'-CCCA terminus in directing
productive end-to-end copying by Q replicase; its importance has
previously been deduced from comparison of the sequences of amplifiable
RNAs (26).
The 3'-most A residue of a CCCA initiation box plays a critical role,
its removal leading to a 5- to 10-fold decrease in end-to-end
transcription (Fig.
5A and experiments not shown). With an initiation
box containing five C residues, the role of a terminal A is less
distinct, its substitution with another base resulting in less
than a
twofold drop in transcription level (Fig.
5B). However,
since Q
RNA
and its related short replicons typically possess
3'-initiation boxes
with three or four C residues (e.g., see Fig.
2A), the 3'-A must be a
critical feature of RNAs replicated in
the cell by Q
replicase. It
is interesting that this A is not
templated by the RNA being
transcribed but rather is added by
the replicase in a step associated
with termination (
25,
36).
Nontemplated 3'-terminal
residues are common among eukaryotic
positive-strand RNA viruses
(
9,
13,
37) and have also been
observed to be necessary
for efficient transcription from the
3' end (see, for example,
references
14 and
31). In cases
where the
viral RdRp itself is responsible for the nontemplated
addition, this
activity could be a viable target for antiviral
drugs.
We have previously reported the inability of Q
replicase to initiate
transcription from a base-paired initiation box (
39),
and
the importance of a 3' end free of secondary structure was
earlier
deduced from the properties common to replicating RNAs
(
4,
26). The results shown in Fig.
6 further demonstrate
the
importance of an accessible initiation site. Although the
3'-CCCA of
E. coli tRNA
His lacking its normal
5'-G (Fig.
6, lane 3) is not base paired,
it is poorly used by Q
replicase. Transcription is increased
by positioning the 3'-A at the
end of a 6-nt unpaired tail (lane
4) and even more by positioning it at
the end of a 9-nt single-stranded
tail (lane 5). These results seem to
indicate that an unstructured
3' end more than 6 nt long is preferred
by the enzyme active site
for initiation at the 3' end. The existence
of base pairing at
the 3' end of Q
RNA (
3) is thought
to be an important contributing
factor in the requirement for host
factor (Hfq) in positive-strand
RNA transcription. 6S RNAs, which have
unstructured 3' ends, are
transcribed independent of the host factor
(
6). Interestingly,
the addition of CC to the 3' end of
Q
RNA largely removes the
Hfq dependence (
28); applying
the conclusions from our study,
we can attribute this effect to a more
extended single-stranded
initiation site and the increased
transcriptional strength derived
from the additional C
residues.
Optimal templates lack CCA or a closely related sequence adjacent
to the 3' end.
The results in Fig. 3 show that predominant
initiation at the 3' end (and consequently end-to-end transcription
that is productive for replication) is not guaranteed by a 3'-CCCA.
When C2-4A is present immediately upstream,
considerable internal initiation can occur: in this position, CCA is
about as potent an initiation site as CCCA is at the 3' end (Fig. 3).
Neither CCA, CCCA, nor CCCCA serve as strong initiation sites when
placed further from the 3' end (Fig. 3, lanes 13 to 15), although we
have observed substantial initiation from a C8A
tract 15 nt from the 3' end of an RNA (39). A priority in
the evolution of a Q replicon would thus clearly be the avoidance of
C2-4A or related C-rich sequences immediately
upstream of the 3' initiation box or at least the placement of such
sequences in base-paired structures. In fact, RNAs amplified by Q
replicase typically lack C-rich sequences in this position (see box #8
in the RNAs listed in Fig. 2A). An exception is Q positive-strand
RNA, in which CCU lies upstream of the 3'-CCCA. We have previously
shown that A, and to a lesser extent G but not U, punctuating a run of
C residues is critical in defining an internal initiation site
(38): that is, initiation will occur from the 3' end of a
run of C-rich pyrimidine residues, either adjacent to a purine
(preferably A) or from the 3' end of the RNA. Thus, the CCUCCCA 3' end
of Q positive-strand RNA should not suffer from excessive internal initiation.
It is interesting that CCA suffices for strong internal initiation from
near the 3' end, whereas an additional C (i.e., CCCA)
is required for a
comparable level of initiation from the 3' terminus.
The
polymerase active site has a clear preference for engaging
a
C-rich initiation box with a short 3' overhang, which presumably
stabilizes the template-enzyme interaction. The recent crystal
structure of the RdRp of the double-stranded RNA bacteriophage
6
(
11), which has revealed strong similarities to the RdRp
of the positive-strand RNA hepatitis C virus, suggests how such
an
overhang may provide initial binding stability. Cocrystallization
of
the RdRp with a DNA template mimic and subsequently with initiating
nucleotide GTP showed that the template initially binds with its
3'-C
positioned in a "specificity pocket" that is one
nucleotide-equivalent
beyond the active site. With GTP present, the
template ratchets
back to position the 3'-C in the active site to
template initiation.
Q
replicase may have an analogous
"specificity pocket," but since
initiation occurs opposite the
3'-penultimate nucleotide, there
would be no need for the template to
ratchet back. For internal
initiation, low-specificity contacts to
3'-proximal nucleotides
could provide sufficient binding stabilization
so that only two
C residues suffice. It is proposed that the
"specificity pocket"
of
6 RdRp can swivel aside to provide a
path for the template
that has already been read to feed through
(
11), and a similar
scheme for Q
replicase could
pertain to internal
initiation.
Can an accessible 3'-CCCA be a sufficiently restrictive
prescription for specific viral amplification in vivo?: the role of
replicase binding sites.
Our studies described above defined a
good template for end-to-end transcription by Q replicase as one
possessing a 3'-CCCA and lacking CC(A/G) immediately upstream. Such a
sequence will appear at the 3' end of an RNA with a probability of ca.
1 in 264. This rather modest specificity is increased to a degree that cannot be quantitated by the requirement that the 3'-CCCA be
accessible. Until in vivo experiments with decoy RNAs are conducted, we
cannot judge whether the transcription dependent on a CCCA initiation box that we have described in this study is sufficiently specific for
viable viral replication. However, because endogenous decoy RNAs that
could serve as nonproductive templates will do so in proportion to
their concentration in the cell, we have perused the range of stable,
abundant E. coli RNAs for the presence of a 3'-CCCA. This
sequence is found only in tRNAHis, which was
demonstrated in Fig. 6 to be a poor template because of the proximity
of the 3'-initiation box to a base-paired helix. tRNAHis should thus only be used to a limited
degree as a template by Q replicase in vivo, and perhaps in practice
not at all, considering that tRNAs are typically engaged in
interactions with aminoacyl-tRNA synthetases, EF-Tu, or the ribosome.
A viable variant of Q
RNA with a 3'-UCCA has been reported
(
28), and we have confirmed that Q
replicase can
effectively
initiate transcription from this sequence, at least when
juxtaposed
next to a CCU upstream sequence (Fig.
7). Among the stable
RNAs
of
E. coli, a 3'-UCCA terminus is only present in
tRNA
Cys (
CCUCCA) and
tRNA
Gly (
GCUCCA; underlined
nucleotides are base paired). Based on the
template activity of
tRNA
His-
5'G (Fig.
6, lane 3), which also has a
4-nt non-base-paired
3' end, these tRNAs should not be efficient
templates.
While the above considerations suggest that transcriptional control
solely by a 3'-initiation box may be feasible, this idea
will certainly
need to be tested experimentally. Specificity could
be readily
augmented with
cis-acting elements that bind replicase,
and
such elements have been reported in the Q
system: the M and
S sites
of Q
positive-strand RNA (
6) and short RNA sequences
that were selected from random sequences by sequential in vitro
binding
(site I and II ligands that are bound by the S1 and EF-Tu
subunits,
respectively, of Q
replicase [
8]). Such binding
sites
could be of benefit in ensuring that replicase will preferentially
interact with viral RNAs in its search for a template, limiting
its
interaction with other cellular RNAs. They could also be beneficial
as
promoters or enhancers, augmenting the transcriptional strength
provided by the initiation boxes described here. No experiments
have
yet tested the contribution of replicase binding sites to
the specific
transcription or replication of Q
RNA in vivo. It
should also be
remembered that other features in addition to an
appropriate initiation
site need to be provided in a successful
replicon. One example is a
high degree of secondary structure
as has been shown necessary for Q
replicons in vivo (
1) and
which has been interpreted to
permit positive and negative strands
to remain separate and to provide
protection against
RNases.
Three Q
replicase binding sites have been implicated in providing
transcriptional strength through in vitro transcription
studies: the M
site in Q
positive-strand RNA (
29) and the site
I and
II ligand RNAs (
8). However, some observations suggest
that
cis-acting sites that tightly bind replicase do not
enhance
transcription or are not needed in RNAs with an accessible
initiation
box. First, as reported in Table
1, we observe comparably
strong
transcription from templates comprised of a 3'-CCCA initiation
box coupled to A-rich sequences that are not tightly bound by
Q
replicase (
6) as from the amplifiable RNA DN3. Second, we
observed no improvement in the transcription of an RNA similar
to CCA9
when the site II ligand sequence was appended to its 5'
end
(
39). Third, no
cis-acting sites common to the
various RNAs
amplifiable by Q
replicase have yet been discerned,
although
there is evidence that the so-called site II on the EF-Tu
subunit
is able to accommodate a rather wide range of single- and
double-stranded
pyrimidine-rich sequences (
23). While such
site II binding may
allow a range of RNAs to bind the replicase, the
loose specificity
could render this site ineffective in discriminating
against nontemplate
RNAs in the cell. The clear requirement for M-site
sequences (
29)
is a peculiarity of Q
positive-strand
RNA that likely relates
to the regulated access of the replicase to an
3'-initiation site
that is deliberately inaccessible in its default
state (due to
base pairing). It has already been convincingly shown
that the
S site functions in the regulation of translation and not
transcription
(
34).
cis-Acting signals that function as replicase binding sites
offer the appealing mechanistic view that productive transcription
occurs after templates attract replicase molecules to the vicinity
of
the active site. On the other hand, transcriptional control
by kinetic
as distinct from binding discrimination has been well
documented in at
least two examples (
16,
35) and could be
the way in which
the "accessibility" of the 3'-initiation box
contributes to
initiation discrimination by Q
replicase. Indeed,
such specificity
control was suggested by Blumenthal (
5), based
on the
observation that template specificity can be overcome by
altering
Mn
2+, glycerol, and GTP concentrations, and that
weak templates required
higher GTP concentrations for initiation. One
can imagine that
the half-life of an initiation site poised in the
active site
awaiting initial phosphodiester bond formation will vary
depending
on the surrounding sequence and the tendency for the
initiation
site vicinity to participate in base pairing and folding. A
tendency
to fold will antagonize active site binding, and bond
formation
will only occur rapidly when the GTP concentration is high
enough
to ensure that the first two GTPs are present in the active site
whenever the unfavored, unfolded conformation of the initiation
site
enters the enzyme active
site.
Further experiments are needed to examine the contribution of the
transcription control mechanisms discussed above to RNA
synthesis by
Q
replicase. A biological system likely benefits
from the use of
diverse mechanisms, and transcription may be controlled
by both
replicase binding and kinetic control strategies. Nevertheless,
the
experiments presented in this study make a strong case that
transcription by Q
replicase is heavily dependent on control
by
3'-CCCA (or closely related) initiation
boxes.
| |
ACKNOWLEDGMENTS |
We are grateful to Michael Farrell of Vysis, Inc., for the
generous gift of Q replicase.
This work was supported by NIH grant GM54610.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR
97331-3804. Phone: (541) 737-1795. Fax: (541) 737-0496. E-mail:
theo.dreher{at}orst.edu.
Technical report 11771 of the Oregon Agricultural Experiment Station.
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Journal of Virology, December 2001, p. 11373-11383, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11373-11383.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
