From the Departments of § Microbiology and
Biochemistry and the ¶ Witebsky Center for Microbial
Pathogenesis and Immunology, State University of New York, School of
Medicine and Biomedical Sciences, Buffalo, New York 14214
Received for publication, December 30, 2002, and in revised form, January 27, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vaccinia virus early gene transcription
termination requires the vaccinia termination factor (VTF), NPH I, a
single stranded DNA-dependent ATPase, the virion form of
RNA polymerase containing the Rap 94 subunit, and the signal UUUUUNU,
which resides in the nascent mRNA, located 30 to 50 bases upstream from
the poly(A) addition site. Evidence indicates that a required
termination factor acts through binding to the UUUUUNU signal. To
further investigate the function of UUUUUNU, the ability of UUUUUNU
containing oligonucleotides to inhibit transcription termination was
tested. A 22-mer RNA oligonucleotide containing a central U9 sequence exhibited sequence and concentration-dependent stimulation of premature
transcription termination and transcript release, in trans.
Activation of premature termination required VTF, NPH I, Rap 94, and
ATP, demonstrating that the normal termination machinery was employed.
Premature termination was not stimulated by RNA harboring a mutant
UUUUUNU, demonstrating specificity. These data are consistent with a
model in which a required termination factor is converted from an
inactive to an active form by binding to a UUUUUNU containing
oligonucleotide. The active termination factor then interacts with the
ternary complex stimulating transcription termination through the
normal mechanism, independent of the nascent mRNA sequence.
Poxviruses are double stranded DNA viruses that replicate in the
cytoplasm of infected cells. To conduct this unusual life cycle,
poxviruses encode the enzymes employed in viral gene transcription, mRNA processing, genome replication, and recombination (1). Vaccinia virus, a member of the poxvirus family, is the strain employed
as smallpox vaccine.
Poxvirus gene expression is divided into three temporal classes that
differ in their promoter sequences and the protein factors employed in
transcription initiation (2). Early genes are transcribed in the virion
core by a virus-encoded multisubunit RNA polymerase (3) that contains
the Rap 94 subunit, the product of the H4L gene (4, 5).
Early messages are capped (6) and polyadenylylated (7) by virus-encoded
enzymes. Shut off of early gene transcription accompanies the onset of
DNA replication. Among the early gene products are proteins that direct
intermediate gene transcription (8, 9). Late gene expression follows
the accumulation of intermediate gene products, including late gene
transcription factors (10). Both intermediate and late gene
transcription require a replicating template and also employ one or
more host-encoded transcription factors (11-13).
Unique of among viral gene classes, early gene transcription is subject
to signal- and factor-dependent transcription termination (14). Termination requires the virion form of RNA polymerase, containing the Rap 94 subunit (15-17), and
VTF1 (18), the vaccinia
termination factor composed of the 97-kDa D1R subunit (19) and the
33-kDa D12L subunit (20). VTF is also the virion mRNA capping
enzyme employed in catalyzing the first three steps in cap formation
(6, 21). In addition, ATPase activity catalyzed by
nucleoside-triphosphate phosphohydrolase I (NPH I), the product of gene
D11L, is essential for transcription termination and
transcript release (22, 23). An interaction between the C-terminal end
of NPH I and the N-terminal end of Rap 94 is required for termination
(15-17, 24). Finally, termination also utilizes the sequence TTTTTNT
present in the gene about 30 to 50 base pairs upstream from the map
position of the early mRNA poly(A) addition site. The signal is
recognized as UUUUUNU in the nascent mRNA (25). One appealing model
proposes that the UUUUUNU signal is recognized by an undefined
essential termination factor and upon binding, this factor initiates
the termination/release sequence of events.
To further investigate the putative UUUUUNU recognition factor we
employed RNA oligonucleotides containing UUUUUNU or a mutated sequence
as potential inhibitors of transcription termination, in
vitro. We hypothesized that binding of the UUUUUNU recognition factor to the UUUUUNU signal in the nascent mRNA would be inhibited in a sequence dependent fashion by the addition of a UUUUUNU containing oligonucleotide, resulting in an inhibition of termination. Analysis of
RNA products synthesized in the presence of different concentrations of
wild type and mutant UUUUUNU containing oligonucleotides showed that
rather than observing an inhibition of termination, we found dramatically enhanced synthesis of truncated transcripts. In addition to the UUUUUNU containing oligonucleotide, the apparent premature termination required VTF, NPH I, Rap 94, and ATP or dATP, demonstrating that the normal viral termination machinery is employed.
Oligonucleotide-dependent premature termination was
independent of the TTTTTNT signal in the template and resulted in
release of the termination products. Importantly, premature termination
required UUUUUNU in the activating oligonucleotide, thus retaining the
essential signal requirement as found in vivo. This
trans-activation of transcription termination demonstrates
that the cis-acting signal present in nascent transcripts can be overridden by an exogenous oligonucleotide, in vitro.
This observation impacts on the mechanism of early gene transcription termination and provides a novel site for the development of potential anti-poxvirus agents.
Cells and Viruses--
Wild type (WT) vaccinia virus strain WR
and the temperature-sensitive mutant virus, tsC50, were propagated in
BSC40 African green monkey cells at 37 °C, or the permissive
temperature for ts mutants, 31 °C, respectively, as described (26,
27). Virus titer was determined by plaque assay on BSC40 cells at the
permissive temperature, 31 °C, and the nonpermissive temperature,
40 °C for tsC50 virus and at 37 °C for the WT virus.
Transcription Extracts--
Transcription extracts were prepared
from virus-infected cells by lysolecithin treatment, as described (23,
28). A549 cells were infected with either WT virus or tsC50 mutant
virus at a multiplicity of infection of 15, at 37 or 31 °C,
respectively. After 24 h, the medium was removed and replaced with
40 °C medium containing 100 µg/ml cycloheximide. After a further
24 h at 40 °C, cells were washed and treated with 250 µg/ml
lysolecithin and extracts were prepared. In the case of the tsC50
virus-infected cells, this procedure permits the initial synthesis of
active NPH I, which is required for intermediate and late gene
expression. After switching to 40 °C, the endogenous NPH I is
inactivated and cycloheximide prevents the synthesis of new protein.
Transcription Termination Assay--
Construction of the
G21(TER29)A78 and G21(TER59)A78 templates was described (29), and
the plasmids containing these sequences were generously provided by Dr.
Stewart Shuman of the Memorial Sloan Kettering Cancer Center.
The G21(TER29)A78 transcription unit contains a synthetic
early promoter fused to a 20-nucleotide G-less cassette, which is
followed by three G residues at positions +21 to +23. RNA synthesis in
the absence of GTP and the presence of 3' O-methyl GTP
yields a 21-base product that can be identified by gel electrophoresis.
A 57-nucleotide A-less cassette lies downstream of the G-less cassette
and flanked at its 3' end by four A residues at positions +78 to +81. A
termination signal, TTTTTTTTT, lies within the A-less cassette,
spanning positions +29 to +37 in the G21(TER29)A78 template. In the
case of the G21(TER59)A78 template, the termination signal, TTTTTTTTT,
lies within the A-less cassette, spanning position +59 to +67. The
biotinylated 324-bp DNA template was amplified by PCR employing a
5'-biotinylated downstream primer and isolated by preparative agarose
gel electrophoresis. The purified DNA fragment was then immobilized to
streptavidin-coated magnetic beads (Dynabeads M280; Dynal) as described
(30).
The bead-bound (B) template (typically, 100 fmol) was first incubated
with 4 µl of C50 or WT virus-infected cell extracts, in the presence
of 1 mM ATP, 10 µCi of [ Transcript Release from the G21 Ternary Complex--
Bead-bound
ternary complexes containing radiolabeled G21 RNA were constructed as
described above using a tsC50 virus-infected cell extract. The ternary
complex was isolated, washed, resuspended, and transcript release from
the paused ternary complex was assessed in the presence or absence of
VTF, NPH I, dATP, and oligonucleotides. After incubation for 10 min at
30 °C, the bound transcript was separated from the free RNA using a
magnet, separated by gel electrophoresis, and analyzed as described
above. To measure the time course of the G21 RNA release, G21 ternary
complexes were prepared as described above using a tsC50 virus-infected
cell extract. Reactions containing ternary complexes, NPH I, VTF, and
dATP were set up and release was begun by the addition of the UUUUUNU
oligonucleotide. At times after adding UUUUUNU, samples were taken and
bound and free G21 RNA were separated using a magnet, and analyzed as
described above.
Prior studies demonstrated that short single stranded
oligonucleotides inhibit overall transcription in vitro,
exhibiting a preferential inhibition of termination (31). The observed inhibition was seen with DNA or RNA and was independent of the UUUUUNU
signal essential for early gene transcription termination. To evaluate
the effect of UUUUUNU containing oligonucleotides on transcription
termination separate from their effect on transcription initiation and
elongation, bead-bound templates were employed. Templates were
described by the Shuman laboratory (29) (Fig. 1B), which permit initiation
of transcription at a strong early promoter and elongation through a
20-nucleotide G-less cassette in the absence of GTP and the presence of
3' O-methyl-GTP, yielding a 21-base RNA product referred to
as G21 RNA. After isolation and washing the bead-bound ternary complex,
elongation in the presence of all four nucleoside triphosphates and
termination factors, NPH I (22, 23) and VTF (18), either full-length transcripts or termination products are formed, which can be separated by gel electrophoresis and observed by autoradiography. Two templates were prepared that differ in the location of the termination signal T9
(Fig. 1B). Ter 29 locates the T9 signal starting at position 29, and Ter 59 has the termination signal starting at position 59. The
position of the termination signal determines the length of the
termination product. Two RNA oligonucleotides were synthesized (Fig.
1A). The UUUUUNU oligonucleotide has a sequence identical to
the nascent transcript synthesized from the Ter 29 template from base
21 to 42. In the mutant oligonucleotide, selected U residues were
changed to A at positions 29, 30, 33, and 36 to yield an altered
termination signal. The essential change is the A at position 33 that
had been shown in the past to generate a sequence that was inactive in
termination (32).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]CTP (800 Ci/mmol), 0.1 mM UTP, and 0.625 mM 3'
O-MeGTP to synthesize the G21 transcript. The ternary
complex was isolated, washed twice with 0.5 ml of transcription salts,
resuspended, and incubated in the presence or absence of VTF that was
preincubated in the presence or absence of the oligo RNA for 10 min on
ice, prior to incubation with the ternary complexes. Whenever
mentioned, the ternary complexes were incubated in the presence or
absence of IgG antibodies directed against different regions of the Rap 94 subunit of the virion RNA polymerase (the H4L protein), prior to
their incubation with VTF and NPH I. Termination was then assessed after elongation of the ternary complex in the presence of 1 mM UTP, 1 mM GTP, 1 mM CTP, and 1 mM ATP. Elongation of the G21 RNA in the presence of all 4 NTPs to the end of the template, would yield a read-through transcript
of about 177 bases in length. UUUUUNU-dependent termination
would yield a termination product of about 70 bases in length. Greater
than 90% of the isolated ternary complexes were routinely elongated in
the second RNA synthesis reaction. RNA products were separated by gel
electrophoresis, observed by autoradiography, and quantified by
densitometry of the exposed film. Termination efficiency was calculated
as the molar ratio of terminated RNA to the sum of read-through and
terminated RNA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
Oligonucleotides and Ter 29 bead-bound
template. A, sequence of the two RNA oligonucleotides
used in this study. One oligonucleotide contains the sequence U9 that
corresponds to the termination signal UUUUUNU. The other is altered to
correspond to a sequence shown to be inactive in transcription
termination (32). B, a map of the bead-bound G21(TER29)A78
DNA template is shown (29). The DNA template is uniquely
biotinylated at the 5' end of the template strand, which anchors
the DNA to streptavidin-coated magnetic beads. The transcription unit
consists of a synthetic early promoter fused to a 20-nucleotide G-less
cassette, which is flanked by a run of three G residues at positions
+21 to +23. A 57-nucleotide A-less cassette was inserted downstream of
the G-less cassette and flanked at its 3' end by four A residues at
positions +78 to +81. A termination signal, TTTTTTTTT, was placed
within the A-less cassette, spanning position +29 to +37.
Arrows represent the RNA products synthesized under various
experimental reaction conditions. The lengths of the RNA products are
noted on the right. B, biotin.
Transcription competent extracts were prepared from cells infected either with wild type virus, or with a mutant virus, tsC50, harboring a ts mutation in the D11L gene, under conditions that yield an extract with reduced NPH I activity. Both extracts are naturally deficient in VTF (23, 33) so maximum termination requires the addition of exogenous VTF, and in the case of C50 extract, both VTF and NPH I must be added. Transcription initiation and elongation to G21 permits the preparation of an isolated ternary complex and effectively separates transcription initiation from termination. The ternary complex can then be incubated with nucleoside triphosphates, oligonucleotides, and termination factors and the radiolabeled nascent RNA allowed either to elongate to the end of the template or to terminate in response to the UUUUUNU signal.
The effect of RNA oligonucleotides containing a wild type or mutant
termination signal on transcription termination was tested. Transcripts
produced in the presence of increasing oligonucleotide concentrations
were observed after separation by gel electrophoresis. In Fig.
2A, lane 2, the
major transcript produced in the absence of added VTF or
oligonucleotide is the full-length read-through transcript (RT). Minor
shorter products (P), including a collection of pause products at the
T9 sequence (30), and the un-elongated G21 RNA (lanes 1 and
10) can be identified. Minimal termination (Term) can be
seen because of the low level of endogenous VTF. When VTF is added,
lane 3, there is a decrease in RT and an increase in Term,
as expected when transcription is conducted with a wild type
virus-infected cell extract (23, 33). Upon the addition of VTF plus
increasing concentrations of a 22-base RNA containing the U9
termination signal (lanes 4-9) one observes a decrease in
Term reflecting an inhibition of transcription termination at the
normal site. Rather than finding a corresponding increase in RT,
however, there is a dramatic increase in the level of short transcripts (PT). The concentration of UUUUUNU oligonucleotide required
for half-maximal PT production is about 2 nM. Titration of
a 22-base RNA possessing a mutant termination signal exhibits a minor
reduction in Term with a minor increase in shorter RNA products
(Fig. 2B). Control studies showed that oligonucleotide addition to the virus-infected cell extracts did not stimulate nuclease
activity (data not shown). These results demonstrate that addition of
an oligonucleotide containing the U9 sequence directs the
signal-dependent stimulation of premature transcription termination, in trans.
|
To determine whether this phenomenon was
template-dependent, the effect of oligonucleotide addition
on transcription termination was evaluated using both the Ter 29 and
Ter 59 templates. In Fig. 3, A
and B, transcription elongation of G21 RNA was carried out in the absence or presence of VTF, and the oligonucleotides. In the
absence of added factors, there is minimal termination (lane 2). Upon the addition of VTF (lane 3), there is an
increase in termination, and as expected, the termination product
produced from the Ter 59 template is longer than that synthesized from Ter 29. Addition of the U5NU oligonucleotide but not the mutant oligonucleotide results in a decrease in the normal termination product
and an increase in the synthesis of the prematurely terminated RNA from
both templates (lanes 4 and 5). Each template
yielded similarly sized premature termination products demonstrating
that the ability of this oligonucleotide to direct the synthesis of short RNA is not template dependent.
|
To assess whether VTF was required for oligonucleotide-directed
stimulation of premature termination, the following study was
conducted. RNA synthesis was carried out in the absence or presence of
VTF and the U5NU oligonucleotide. In Fig.
4A, lane 1, one
observes primarily the synthesis of read-through RNA in the absence of
added factors. When VTF is added (lane 3), the Term RNA
accumulates. If the U5NU oligonucleotide is added in the absence of
VTF, there is no change (lane 2). However, if the U5NU RNA
is added in the presence of VTF (lane 4) the accumulation of
the premature termination products is observed. When the VTF concentration is increased in the presence of the U5NU RNA (Fig. 4B) there is a decrease in the synthesis of the read-through
RNA and a marked increase in the synthesis of premature termination products. At high levels of VTF there is a reappearance of a low level
of the normal termination product (Term), whereas the level of
prematurely terminated RNA varies little. The normal termination products are formed from ternary complexes that escape UUUUUNU-directed premature termination.
|
The requirement for NPH I for U5NU
oligonucleotide-dependent generation of premature
termination products was evaluated through the use of a transcription
competent extract prepared from tsC50-infected cells, possessing
reduced NPH I (23, 33). In Fig.
5A, lane 1, one can
observe the expected RNA products in the absence of added factors. When
the U5NU oligonucleotide (lane 2) is added, there is no
change. When VTF is added (lane 3) there is a minor increase
in the normal termination product (Term) because of a low level of
endogenous NPH I in the tsC50 virus-infected cell extract. When VTF is
added in the presence of U5NU (lane 4), there is a minor
increase in premature termination products. If NPH I is added in the
absence of VTF (lane 5) there is little stimulation of
termination or of the level of prematurely terminated RNA in the
presence of added U5NU (lane 6) because of the low level of VTF in the infected cell extract. However, if NPH I and VTF are added,
normal termination is observed (lane 7). If U5NU RNA is added in addition, one observes the stimulation of premature
termination (lane 8). As in the case of VTF, addition of
increasing levels of NPH I results in enhanced premature termination,
Fig. 5B.
|
Early gene transcription termination was shown to require the
N-terminal region of the Rap 94 subunit, the product of gene H4L (15, 16, 24). Antibodies that bind to the N-terminal end
of Rap 94 prevent termination in vitro. A requirement for Rap 94 for premature termination was tested (Fig.
6). In this case, a tsC50-infected cell
extract was employed that was deficient in both VTF and NPH I activity.
In the absence of added factors, the read-through RNA and pause
products were observed (lane 2). Addition of VTF alone had
little effect (lane 3) because the extract has low NPH I
activity. Addition of VTF and NPH I resulted in the appearance of the
normal termination product, Term (lane 4). Addition of
UUUUUNU along with VTF and NPH I (lane 5) caused the accumulation of the PT products. As shown previously (16),
preincubation of the isolated ternary complexes with antibodies raised
against the N-terminal but not the C-terminal region of Rap 94 inhibited normal transcription termination (compare lanes 8 and 9). However, pretreatment of the ternary complexes with
antibodies raised against the N-terminal region of Rap 94 but not those
derived from the C-terminal region of Rap 94 prevented UUUUUNU
oligonucleotide stimulation of premature termination (compare
lanes 6 and 7).
|
Early gene transcription termination requires energy generated by NPH
I-catalyzed ATP hydrolysis (22, 23). To evaluate the energy requirement
for U5NU-stimulated premature termination the standard transcription
protocol was modified. Radiolabeled G21 RNA containing ternary
complexes were prepared. Elongation through the A-less cassette to A78
was carried out in the absence of added ATP, and the presence of dATP
or cordycepin triphosphate. In Fig.
7A, lanes 1 and
2, display RNA was made in the presence of VTF, and the
presence or absence of U5NU RNA. One observes the read-through RNA
because of low levels of contaminating ATP. A prominent RNA product
labeled A78 is formed because of pausing near A78 in the absence of
added ATP. A78 migrates near the normal termination product, Term,
which would be minimal in this assay because of the low level of ATP in
these reactions (30). When cordycepin triphosphate is added along with
VTF (lanes 3, 4, 7, and 8),
one observes a failure to synthesize the read-through RNA because of
the incorporation of the chain terminating base at A78. Addition of
U5NU (lanes 4 and 8) stimulates premature termination indicating that cordycepin triphosphate can serve as an
energy source. If dATP is added in the absence of cordycepin triphosphate (lane 5) one observes a failure to synthesize
the read-through product. When U5NU is added (lane 6),
premature termination products are observed demonstrating that dATP can
serve as the energy source required for premature transcription
termination. Addition of both cordycepin triphosphate and dATP has no
additional effect (lanes 7 and 8).
|
To determine whether the newly synthesized RNA products are released from the ternary complexes, after elongation, bound and free RNA were separated and analyzed by gel electrophoresis. In Fig. 7B one observes that in the absence of added ATP, the RNA remains bound in the ternary complex whether U5NU is added or not (lanes 1-4). If an energy source is provided in the form of cordycepin triphosphate or dATP, the majority of the RNA products are released, A78, Term, and the premature termination products (lanes 5-16). Release occurs in the absence of added UUUUUNU oligonucleotide because of the fact that the Ter 29 template was employed that directs the synthesis of nascent RNA with an available UUUUUNU signal. Addition of the UUUUUNU oligonucleotide stimulates release in each case.
To ensure that the endogenous U5NU does not contribute to the
oligonucleotide-dependent stimulation of transcript release and to determine whether an extended RNA 5' tail is needed, the ternary
complex stalled at position G21 was employed in the transcript release
assay because this complex was certain to possess a short nascent
transcript that lacks U5NU. Ternary complexes were constructed using a
tsC50 mutant virus-infected cell extract. Incubations of the isolated,
washed ternary complexes were conducted in the absence or presence of
NPH I, VTF, and dATP, in the absence or presence of oligonucleotides
that contained either a wild type or a mutated UUUUUNU termination
signal (Fig. 8A). When dATP is omitted (lanes 1-4) there is little release of G21 RNA
because of the lack of an energy source. If VTF is omitted (lanes
5-8) there is a modest stimulation of release (16%) when UUUUUNU
RNA is added because of the low level of endogenous VTF in this assay. The mutant oligonucleotide has a much lower effect on release (lanes 7 and 8). If NPH I is omitted (lanes
11-14) a low level of transcript release is observed that is
higher (22%) when the UUUUUNU oligonucleotide is added rather than the
mutant oligonucleotide (10%). If oligonucleotide is not added
(lanes 9 and 10) only 20% release is observed.
However, in the presence of VTF, NPH I, and dATP a high level (57%) of
G21 RNA release can be observed in the presence of the UUUUUNU
oligonucleotide (lanes 15 and 16). Only
background release is observed when the mutant oligonucleotide is added
(lanes 17 and 18). Release is rapid achieving
near maximal release within 30 s after initiating the reaction
(Fig. 8B). These results demonstrate that UUUUUNU stimulates
a release factor that does not require ongoing transcription, is
independent of the UUUUUNU signal in the nascent RNA, and does not
require an extensive RNA 5' tail.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A current model for termination of vaccinia virus early gene transcription describes the interplay of the nascent mRNA, the virion RNA polymerase, VTF, NPH I, and the template DNA (34). NPH I is a single stranded DNA-dependent ATPase (35, 36) that provides energy needed for transcript release (22, 23). The requirement for single stranded DNA for adoption of the active conformation indicates that NPH I is able to bind to DNA in an accessible region in the transcription bubble. Furthermore, studies demonstrated that the C-terminal end of NPH I must bind to the N-terminal domain of the RNA polymerase Rap 94 subunit (15, 16, 24), the product of gene H4L. Loss of NPH I binding or antibody binding to Rap 94 prevents termination, in vitro. A role for VTF, the first termination factor to be identified (18), has not been defined. However, VTF is clearly required for transcript release, in vitro (29).
The sequence UUUUUNU located upstream from the poly(A) addition site is essential for termination in vitro (32). Alteration of any U residue results in a reduction in termination efficiency. The UUUUUNU sequence resides 30 to 50 bases upstream from the 3' end (37). This location places the UUUUUNU signal outside of the RNA polymerase in a region of the nascent mRNA that is not protected by the RNA polymerase from nuclease digestion (29). These two facts support a model in which the UUUUUNU sequence binds to an essential termination factor as a required step in transcription termination. Although VTF is a likely candidate, to date, there is no evidence to support this proposition.
These studies were undertaken to test the hypothesis that a UUUUUNU recognition factor binds to UUUUUNU and participates in early gene transcription termination. Addition of exogenously added RNA containing a U9 sequence was expected to inhibit normal transcription termination by binding to the termination factor and preventing its association with the UUUUUNU sequence in the nascent transcript. A sequence-dependent reduction in the normal termination product was seen, as anticipated. Unexpectedly, the accumulation of short transcripts was also observed. Premature termination was not determined by the template because it was found when either the Ter 29 or Ter 59 templates were used. In addition, premature termination required not only the sequence UUUUUNU in the activating oligonucleotide but also VTF, NPH I, the N-terminal domain of Rap 94 and dATP, demonstrating that the normal transcription termination machinery was employed. Finally, UUUUUNU RNA was shown to stimulate the release of the G21 transcript from the isolated ternary complex. Because it was shown that RNA polymerase protects 18 bases of the nascent transcript from nuclease digestion, and the G21 RNA is not available for cap formation, in vitro (29, 38), UUUUUNU oligonucleotide-dependent transcript release must not require an extensive interaction with the 5' tail of the target RNA.
The premature termination products vary somewhat in length from experiment to experiment. This is because of the variable efficiency of release factor stimulation. Although release of G21 RNA in the absence of elongation is efficient (Fig. 8), in practice, there is a competition between elongation and release, in vitro.
Hybrid formation between the U9 oligonucleotide and either the nascent transcript or the template is not likely to contribute to premature termination. The nascent transcript lacks an oligo A sequence that would permit hybrid formation. The template strand has an A9 sequence, which could be a potential target for hybrid formation. However, G21 RNA is efficiently released from a ternary complex that has not reached the A9 sequence in the Ter 29 template. In addition, efficient premature termination is also observed with the Ter 59 template where the A9 sequence lies in a different sequence context. Finally a DNA oligonucleotide of the same sequence, where T is substituted for U, which would be fully capable of hybrid formation, has no effect on premature termination, in vitro.
The most parsimonious model proposes that a UUUUUNU containing RNA oligonucleotide can bind to an inactive transcription termination factor and convert it to the active form. When activated, the termination factor initiates a sequence of events that results in release of the nascent RNA from the ternary complex. Normally, the activating UUUUUNU is present in the nascent RNA. This demonstrates that the newly formed transcript must be scanned by the recognition factor as transcription elongation proceeds. Because the termination occurs within 30 bases of the UUUUUNU sequence, scanning must be quite efficient. When the UUUUUNU signal is added in trans, however, the nascent transcript is disregarded and efficient transcript release is obtained without concomitant transcription elongation. Titration of the UUUUUNU containing RNA oligonucleotide exhibits a half-activation concentration of about 2 nM demonstrating a remarkably strong binding to the UUUUUNU recognition factor.
The identity of the UUUUUNU recognition factor remains to be determined. VTF is a likely candidate because it is known to be required but lacks a termination function at this time. VTF is known to catalyze the first three steps in mRNA cap formation (6, 21), and in this capacity, VTF is an RNA-binding protein. At least two RNA interaction sites have been identified: one in the N-terminal 250 amino acids of the large D1R subunit (39, 40) and the other in the C-terminal end of D1R, in the methyltransferase domain. Although VTF was reported to exhibit a modest binding preference for poly(U) (41), it is not clear whether this preferential affinity for Us is related to transcription termination, mRNA cap formation, or intermediate gene transcription, other known functions of VTF. It is important to keep a broad perspective in the absence of conclusive data. UUUUUNU could be recognized by an unknown termination factor that acts in trans. Alternatively, one of the components of the transcription complex, perhaps an RNA polymerase subunit, might bind to UUUUUNU and stimulate termination. Studies are underway to identify the UUUUUNU binding component and elucidate its role in transcription termination.
It is interesting to note that in our assays, 20-30% of the ternary
complexes are unable to terminate in response to added VTF and ATP.
Evidence indicates that these same ternary complexes are not subjected
to UUUUUNU oligonucleotide-directed premature termination because the
level of read-through transcript is relatively constant throughout the
UUUUUNU titration. The premature termination products accumulate at the
expense of the normal terminated RNA. Similarly, a fraction of the
ternary complexes formed at A78 do not respond to
factor-dependent transcript release (29). It is likely that
these complexes are missing an essential component that is not replaced
simply by further addition of excess VTF or NPH I. UUUUUNU directly
stimulates RNA release from about 80% of the G21RNA ternary complexes
(Fig. 8B). Moreover, addition of UUUUUNU oligonucleotide to
an ongoing transcription reaction results in near complete release of
the nascent transcripts (Fig. 7B). These observations
indicate that the ternary complexes are heterogeneous in regard to
their ability to respond to normal and UUUUUNU-directed transcription
termination. An understanding of the differences among the ternary
complexes would provide information important to our understanding of
the mechanism of early gene transcription termination.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Stewart Shuman for valuable contributions to this research.
![]() |
FOOTNOTES |
---|
* This work was supported by NIAID National Institutes of Health Grant RO1-AI 43933.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Microbiology
Department, 138 Farber Hall, SUNY School of Medicine, 3435 Main St., Buffalo, NY 14214. Tel.: 716-829-3262; Fax: 716-829-2169; E-mail: eniles@buffalo.edu.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M213263200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: VTF, vaccinia termination factor; NPH I, nucleoside-triphosphate phosphohydrolase I; Rap 94, RNA polymerase-associated protein 94 kDa; WT, wild type; RT, read-through transcript; Term, termination product; PT, premature termination; U9P, P, pause products; A78, A78 RNA; G21, G21 RNA; U, U5NU oligo; M, mutant U5NU oligo.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Moss, B. (2001) in Poxviridae: The Viruses and Their Replication: Virology (Knipe, D. M. , Howley, P. M. , Griffin, D. E. , Martin, M. A. , Lamb, R. A. , Roizman, B. , and Strauss, S. E., eds), Vol. 2 , pp. 2849-2883, Lippincott-Raven, Philadelphia |
2. |
Moss, B.,
Ahn, B. Y.,
Amegadzie, B.,
Gershon, P. D.,
and Keck, J. G.
(1991)
J. Biol. Chem.
266,
1355-1358 |
3. |
Baroudy, B. M.,
and Moss, B.
(1980)
J. Biol. Chem.
255,
4372-4380 |
4. | Ahn, B. Y., and Moss, B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3536-3540[Abstract] |
5. | Kane, E., and Shuman, S. (1992) J. Virol. 66, 5752-5762[Abstract] |
6. | Wei, C. M., and Moss, B. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 3014-3018[Abstract] |
7. | Kates, J. R., and Beeson, J. (1970) J. Mol. Biol. 50, 19-33[Medline] [Order article via Infotrieve] |
8. | Vos, J. C., Sasker, M., and Stunnenberg, H. G. (1991) EMBO J. 10, 2553-2558[Abstract] |
9. |
Rosales, R.,
Harris, N.,
Ahn, B. Y.,
and Moss, B.
(1994)
J. Biol. Chem.
269,
14260-14267 |
10. | Keck, J. G., Baldick, C. J., Jr., and Moss, B. (1990) Cell 61, 801-809[Medline] [Order article via Infotrieve] |
11. |
Gunasinghe, S. K.,
Hubbs, A. E.,
and Wright, C. F.
(1998)
J. Biol. Chem.
273,
27524-27530 |
12. | Rosales, R., Sutter, G., and Moss, B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3794-3798[Abstract] |
13. |
Wright, C.,
Oswald, B.,
and Dellis, S.
(2001)
J. Biol. Chem.
276,
40680-40686 |
14. | Rohrmann, G., Yuen, L., and Moss, B. (1986) Cell 46, 1029-1035[Medline] [Order article via Infotrieve] |
15. |
Mohamed, M. R.,
and Niles, E. G.
(2000)
J. Biol. Chem.
275,
25798-25804 |
16. |
Mohamed, M. R.,
and Niles, E. G.
(2001)
J. Biol. Chem.
276,
20758-20765 |
17. | Mohamed, M. R., Christen, L., and Niles, E. G. (2002) Virology 299, 142-153[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Shuman, S.,
Broyles, S. S.,
and Moss, B.
(1987)
J. Biol. Chem.
262,
12372-12380 |
19. | Morgan, J. R., Cohen, L. K., and Roberts, B. E. (1984) J. Virol. 52, 283-297 |
20. | Niles, E. G., Lee-Chen, G. J., Shuman, S., Moss, B., and Broyles, S. S. (1989) Virology 172, 513-522[Medline] [Order article via Infotrieve] |
21. | Ensinger, M. J., Martin, S. A., Paoletti, E., and Moss, B. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 2525-2529[Abstract] |
22. |
Deng, L.,
and Shuman, S.
(1998)
Genes Dev.
12,
538-546 |
23. | Christen, L. M., Sanders, M., Wiler, C., and Niles, E. G. (1998) Virology 245, 360-371[CrossRef][Medline] [Order article via Infotrieve] |
24. | Piacente, S. C., Christen, L. M., Mohamed, M. R., and Niles, E. G. (2002) Virology, in press |
25. |
Shuman, S.,
and Moss, B.
(1989)
J. Biol. Chem.
264,
21356-21360 |
26. | Condit, R. C., and Motyczka, A. (1981) Virology 113, 224-241[Medline] [Order article via Infotrieve] |
27. | Condit, R. C., Motyczka, A., and Spizz, G. (1983) Virology 128, 429-443[Medline] [Order article via Infotrieve] |
28. | Condit, R. C., Lewis, J. I., Quinn, M., Christen, L. M., and Niles, E. G. (1996) Virology 218, 169-180[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Deng, L.,
Hagler, J.,
and Shuman, S.
(1996)
J. Biol. Chem.
271,
19556-19562 |
30. |
Hagler, J.,
Luo, Y.,
and Shuman, S.
(1994)
J. Biol. Chem.
269,
10050-10060 |
31. | Christen, L., Sanders, M., and Niles, E. G. (1999) Biochemistry 36, 8072-8089[CrossRef] |
32. | Yuen, L., and Moss, B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6417-6421[Abstract] |
33. | Condit, R. C., Xiang, Y., and Lewis, J. I. (1996) Virology 220, 10-19[CrossRef][Medline] [Order article via Infotrieve] |
34. | Condit, R. C., and Niles, E. G. (2002) Biochem. Biophys. Acta 1577, 325-336[Medline] [Order article via Infotrieve] |
35. |
Paoletti, E.,
and Moss, B.
(1974)
J. Biol. Chem.
249,
3281-3286 |
36. | Paoletti, E., Rosemond-Hornbeak, H., and Moss, B. (1974) J. Biol. Chem. 249, 273-280 |
37. | Yuen, L., and Moss, B. (1986) J. Virol. 60, 320-323[Medline] [Order article via Infotrieve] |
38. | Hagler, J., and Shuman, S. (1992) Science 255, 983-986[Medline] [Order article via Infotrieve] |
39. |
Higman, M. A.,
Christen, L. A.,
and Niles, E. G.
(1994)
J. Biol. Chem.
269,
14974-14981 |
40. |
Myette, J. R.,
and Niles, E. G.
(1996)
J. Biol. Chem.
271,
11936-11944 |
41. |
Luo, Y.,
and Shuman, S.
(1993)
J. Biol. Chem.
268,
21253-21262 |