The HIV-1 inducer of short transcripts (IST) is
an unusual promoter element that activates the synthesis of short
transcripts from the HIV-1 promoter as well as from heterologous
promoters. While the DNA sequences constituting IST have been
characterized in some detail, little is known about the biochemical
mechanisms underlying IST activity. Here, we describe a cell-free
transcription assay that faithfully reproduces the synthesis of
IST-dependent HIV-1 short transcripts. As in
vivo, formation of these short transcripts requires a functional
IST element and is repressed in the presence of the viral
trans-activator Tat. Short transcript and full-length
transcript synthesis respond differently to variations in several
reaction parameters, suggesting that the short and full-length
transcripts are synthesized by transcription complexes with distinct
biochemical properties. In particular, short transcript synthesis is
resistant to the action of
5,6-dichloro-1-
-D-benzimidazole, an inhibitor of
transcript elongation. Formation of transcription complexes directed by
the IST element may, therefore, not require the activity of a factor
inhibited by 5,6-dichloro-1-
-D-benzimidazole, such as
the TFIIH-associated or pTEFb kinases.
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INTRODUCTION |
The HIV-1 promoter directs the synthesis of two classes of RNAs
that are initiated at the same transcription start site: full-length transcripts extending to the 3'-end of the transcription unit, and
short, nonpolyadenylated RNAs of approximately 55-65 nucleotides in
length. The relative levels of these two classes of transcripts are
modulated by the viral transcriptional activator Tat, which activates
synthesis of the full-length transcripts but down-regulates synthesis
of the short transcripts (1-3). While full-length transcripts are
probably synthesized by elongation-competent transcription complexes
capable of traversing the entire transcription unit, short transcripts
have been hypothesized to result from a separate class of transcription
complexes incapable of efficient transcript elongation (2, 4). The
biochemical nature of these putative elongation-incompetent
transcription complexes is unknown, although the observation that
mutations in the TATA box-binding protein have similar effects on the
synthesis of short and full-length transcripts suggests that
elongation-competent and elongation-incompetent transcription complexes
do not differ in their use of TATA box-binding protein (5). In
vivo, formation of the short transcripts depends on an unusual
promoter element termed the inducer of short transcripts (IST)1 (2). Mutations in IST
preferentially diminish the synthesis of the short transcripts but have
a much lesser effect on the synthesis of the full-length transcripts.
IST is a bipartite DNA element located predominantly downstream of the
start site of transcription. Its major determinants are contained
within positions
5 to +26, while sequences between +40 and +66
further augment its activity (4, 6). Whereas the cis-acting
sequences involved in IST function have thus been characterized in some
detail, nothing is known about the mechanism of IST action. Here, we
describe the establishment of a cell-free transcription assay that
faithfully reproduces the synthesis of IST-directed HIV-1 short
transcripts, as judged by the following two criteria: their synthesis
(i) depends on an intact IST element, and (ii) is reduced upon the
addition of the Tat protein. We show that synthesis of the short
transcripts is inhibited by low concentrations of
-amanitin,
indicating that they are the products of RNA polymerase II. Unlike
formation of the full-length transcripts, formation of the short
transcripts is resistant to addition of the nucleoside analog DRB, an
inhibitor of various cellular protein kinases, suggesting that the
short and long transcripts are synthesized by different RNA polymerase II transcription complexes.
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EXPERIMENTAL PROCEDURES |
Plasmids--
Plasmid pU3RIII/pUC119 (generously provided by M. Laspia, Dartmouth Medical School) contains HIV-1 sequences from
642
to +82 fused to the chloramphenicol acetyltransferase gene and is identical to pU3RIII (7) except that the vector backbone is derived
from pUC119. The plasmids used in the cell-free transcription assays,
pHIV-1/R/ML and derivatives, were constructed as follows. In the first
step, the construct pU3RIII/pUC119, which contains two
HindIII sites, was subjected to a partial digestion with
HindIII, the ends were filled-in with the Klenow fragment of
Escherichia coli DNA polymerase I, linear molecules were
isolated on a gel and religated. A clone containing a single
HindIII site at position +78 relative to the HIV-1
transcription start site was isolated and named
pU3RIII/pUC119/HindIII
. In the second step,
the construct pU3RIII/pUC119/HindIII
, which
contains two ScaI site and a unique HindIII site,
was subjected to partial digestion with ScaI, followed by
complete digestion with HindIII and phosphatase treatment.
Linear molecules cleaved at the ScaI site at position
141
relative to the HIV-1 transcription start site and at the
HindIII site at position +78 relative to the HIV-1
transcription start site were isolated. This vector was ligated with a
fragment generated by polymerase chain reaction from the pHIV-1/R
template (4) and extending from the ScaI site at position
141 to the HindIII site at position +78. The resulting
construct, pHIV-1/R/ML, was similar to the pU3RIII/pUC119 construct,
except that the HIV-1 sequences contained an engineered XhoI
site at position
10, and a point mutation at position +77 that
disrupts the polyadenylation signal. This construct contains unique
XhoI and AflII sites at positions
10 and +64,
respectively, relative to the HIV-1 transcription start site. The
derivatives of pHIV-1/R/ML were generated by replacing the
XhoI-AflII fragment extending from
10 to +64 by
the corresponding XhoI-AflII fragments derived
from the various pHIV-1/R derivatives.
Template plasmids for the synthesis of antisense riboprobes were
constructed as follows. pHIV-1/R was digested with HindIII, which cleaves just proximal to a bacteriophage T3 promoter oriented in
an antisense direction relative to the HIV-1 promoter, and the ends
were filled in with the Klenow fragment of E. coli DNA polymerase I. The linear plasmid was then digested with
XhoI, which cuts at position
9 with respect to the HIV-1
transcription start site. Plasmids pHIV-1/R/ML or ABC/ML were cleaved
with EcoRI at position +329 in the chloramphenicol
acetyltransferase coding sequences and the ends were filled in with the
Klenow fragment of DNA polymerase I. Subsequent digestion with
XhoI liberated a 334-base pair fragment which was ligated
into the XhoI/HindIII cut pHIV-1/R described
above, resulting in plasmids pHIV-1/ML/T3/
Eco and pABC/ML/T3/
Eco.
Fig. 1B depicts maps of the above constructs and the
position of the antisense RNA probe with respect to the HIV-1
transcription unit.
Cell-free Transcription Assay--
Plasmid templates were
linearized at the NcoI site at position +630 with respect to
the HIV-1 transcription start site, phenol and phenol/chloroform
extracted, ethanol precipitated, and dissolved in deionized sterile
water at a concentration of 250 ng/µl. Unless otherwise indicated,
transcription reactions (20 µl) contained 250 ng of linearized
plasmid, 7.5 mM MgCl2, 50 mM KCl, 2 mM dithiothreitol, 10 mM HEPES-KOH (pH 7.9),
3.75 mM creatine phosphate, 250 ng of poly(dG-dC)·poly(dG-dC), 250 µM each ATP, CTP, and GTP,
7.5 µM UTP, 10 µCi (165 nM) of
[
-32P]UTP (1 µl of 3000 Ci/mmol in 50 mM
Tricine, pH 7.6, NEN Research Products), and 10 µl of HeLa cell
nuclear extract (80-120 µg of protein). Reactions were incubated at
30 °C for 60-75 min with no preincubation or presynthesis.
Transcription was terminated by the addition of 200 µl of stop buffer
(330 mM NaCl, 0.5% SDS, 10 mM Tris-HCl (pH
7.5), 10 µg/100 µl of total yeast RNA, 50 µg/100 µl of
proteinase K), and, after a further incubation of 10 min at room
temperature, nucleic acids were isolated by phenol/chloroform extraction and ethanol precipitation. Precipitated nucleic acids were
then redissolved in formamide loading buffer, resolved by electrophoresis on denaturing 6% polyacrylamide gels, and visualized by autoradiography (Kodak X-Omat AR) for 2-24 h. In some experiments,
-amanitin or 5,6-dichloro-1-
-D-benzimidazole (DRB)
were added to transcription reactions before the initiation of RNA
synthesis, at the concentrations indicated in the figure legends. When
indicated, 0.5 µl of a buffer containing 250 ng/µl Tat protein, 50 mM KCl, 10 mM HEPES-KOH (pH 7.9), 1 mM EDTA, and 5 mM dithiothreitol was added at
the beginning of the transcription reaction (kindly provided by M. Laspia, Dartmouth School of Medicine).
DRB was kept at
20 °C as a 1.6 mM solution in 10%
ethanol, 10 mM HEPES-KOH (pH 7.9). Immediately prior to
use, an aliquot was placed in a 65 °C water bath until it was
completely dissolved and then kept in a 37 °C water bath to prevent
precipitation. Two µl was added to the transcription reaction prior
to the addition of template, resulting in a final concentration of 160 µM. The Ad2 VAI gene was transcribed as described
previously (8).
Synthesis of Unlabeled Antisense Riboprobes--
Unlabeled
antisense riboprobes were synthesized in vitro with T3 RNA
polymerase (Boehringer Mannheim) as follows. Template plasmids
pHIV-1/ML/T3/
Eco or pABC/ML/T3/
Eco were linearized at the Asp-718
restriction site at position
146 with respect to the HIV-1
transcription start site, phenol/chloroform extracted, ethanol
precipitated, and redissolved in sterile deionized water. Two µg of
template were then incubated at 37 °C for 1 h in 40 µl of
in vitro RNA synthesis reaction containing 40 units of
bacteriophage T3 RNA polymerase, 4 mM each nucleotide
triphosphate, 26 mM MgCl2, 40 mM
Tris, pH 7.5, 2 mM spermidine, 10 mM NaCl, and
40 units of RNasin (Promega). The resulting unlabeled RNAs were
phenol/chloroform extracted, ethanol precipitated, and resolved by
electrophoresis on a denaturing 6% (w/v) polyacrylamide gel. RNA bands
of the expected electrophoretic mobility were visualized by UV
shadowing, excised from the gel, and eluted into a buffer containing
330 mM NaCl and 0.5% SDS by shaking overnight at 37 °C.
They were then phenol/chloroform extracted, ethanol precipitated, and
redissolved in sterile deionized water at a concentration of 250 ng/µl.
Nuclease Protection Assay--
Phenol/chloroform-extracted and
ethanol-precipitated nucleic acids from a cell-free transcription
reaction were incubated at 37 °C for 10 min in a buffer containing
50 mM NaAc (pH 7.0), 10 mM MgCl2, 2 mM CaCl2, 5 units of RNasin (Amersham), and 5 units of DNase I to remove the template DNA. The reaction was then
phenol/chloroform extracted, ethanol precipitated, redissolved in 40 µl of hybridization buffer containing 80% formamide, 400 mM NaCl, 40 mM PIPES-KOH (pH 6.4), 1 mM EDTA, and 250 ng of unlabeled antisense riboprobe, and
the nucleic acids were then hybridized at 46 °C overnight. RNase T1
digestion was performed by addition of 300 µl of a buffer containing
330 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 3000 units of RNase T1 and subsequent
incubation at 30 °C for 60 min. The RNase was then inactivated by
addition of SDS to a final concentration of 0.5% and 60 µg of
proteinase K, and incubation at 37 °C for 15 min. Reactions were
phenol/chloroform extracted, ethanol precipitated, and protected RNAs
were resolved by electrophoresis on a denaturing 6% (w/v)
polyacrylamide gel and visualized by autoradiography.
RNA Stability Assay--
Plasmids pET7/R and pET7/ABC contain
sequences from +1 to +82 of either pHIV-1/R or msABC (4), respectively,
cloned downstream of the bacteriophage T7 promoter into the
StuI site of plasmid pET7 (9). Internally labeled short
transcripts carrying wild-type HIV-1 sequences from +1 to +68 or the
same sequence segment containing the ABC mutations were synthesized as
follows. The template plasmids were linearized at the AflII
site at position +64 of the HIV-1 insert, phenol/chloroform extracted,
ethanol precipitated, and redissolved in sterile deionized water at a
concentration of 250 ng/µl. 250 ng of template were then used in a
standard bacteriophage T7 RNA polymerase (Boehringer Mannheim) in
vitro RNA synthesis reaction according to the recommendations of
the manufacturer, except that the reaction contained 6.25 µM [
-32P]CTP (NEN Research Products, 800 Ci/mmol) and 100 µM unlabeled CTP. The resulting
transcripts were purified as described above, quantitated by Cerenkov
counting in a scintillation counter, and redissolved in sterile
deionized water at a concentration of 20,000 counts/µl. 40,000 counts
of radiolabeled wild-type or ABC short transcripts were then added to
reactions that were identical to a standard cell-free transcription
reaction except that they did not contain
[
-32P]UTP.
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RESULTS |
Synthesis of IST-dependent Short Transcripts in a
Cell-free Transcription Assay--
We have previously characterized
mutations within the HIV-1 promoter region that affect preferentially
the synthesis of short transcripts, full-length transcripts, or both
(4). To determine whether we could obtain short transcript synthesis
in vitro, we compared the RNA products generated by a
battery of such mutants in a cell-free system. However, we detected
only weak transcriptional signals when a HeLa cell nuclear extract was
programmed with the constructs we had used before in our in
vivo studies (pHIV-1/R and its mutant derivatives, data not
shown). We therefore subcloned HIV-1 sequences containing the wild-type
or mutant IST sequences (from
141 to +78) into an HIV-1 construct
that had been used successfully for in vitro transcription
studies (7), thus creating the construct pHIV-1/R/ML and its
derivatives, whose sequences in the
30 to +92 region are shown in
Fig. 1A. These constructs contain HIV-1 sequences from
642 to +82 relative to the transcription start site fused to the chloramphenicol acetyltransferase coding sequences. The polyadenylation signal starting at position +74, which
is not used in the 5'-long terminal repeat in vivo, was inactivated by a point mutation at position +77 (underlined
in Fig. 1A) to avoid the possibility of artifactual
polyadenylation of transcripts in vitro. A schematic of
pHIV-1/R/ML is depicted in Fig. 1B.

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Fig. 1.
A, sequences of the wild-type and mutant
HIV-1 templates for in vitro transcription. The
transcription start site is labeled +1. The
underlined sequences between 10 and 5 correspond to a
XhoI site that was introduced by point mutations, and the
underlined nucleotide at position +77 corresponds to a point
mutation introduced to debilitate the polyadenylation signal. The
location of the IST element is indicated by brackets. The
ability of each mutant to synthesize short transcripts in a
transfection assay (3, 4) is indicated on the right (+, no
selective defect in short transcript synthesis; +/ , partial defect in
short transcript synthesis; , selective defect in short transcript
synthesis). B, schematic of the wild-type template plasmid
used in the cell-free transcription assay (pHIV-1/R/ML), the template
plasmid used for in vitro synthesis of the wild-type
antisense riboprobe (pHIV-1/ML/T3/ Eco), the location of the
antisense riboprobe with respect to the transcription unit in
pHIV-1/R/ML, and the protected transcripts. The arrow in
pHIV-1/R/ML denotes the start site of transcription. The
arrow in pHIV-1/ML/T3/ Eco refers to the
bacteriophage T3 promoter in antisense orientation used to synthesize
the antisense riboprobe.
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The pHIV-1/R/ML plasmid and mutant derivatives were linearized with
NcoI at position +630 downstream of the HIV-1 transcription start site, and were then used to program a HeLa cell nuclear extract
in the presence of radiolabeled uridine triphosphate. The RNA products
were fractionated on a sequencing gel, and the results are shown in
Fig. 2. As typically observed with this
type of assay, a number of background bands resulting from
transcription initiation at cryptic promoters within vector sequences
and from end labeling of endogenous RNAs found in the nuclear extract
were detected (e.g. bands around the 110 and 67 nucleotide
markers). In addition, we detected two classes of specific transcripts: transcripts of about 630 nt, the length expected for full-length run-off transcripts, and short transcripts of about 83 and 85 nt in
length (Fig. 2, lane 3, bands labeled FL and
st, respectively. Note that only the sections of the gel
containing the bands of interest are shown; a full gel is shown in Fig.
5A.) The appearance of these transcripts depended on the
presence of an intact HIV-1 TATA box, because they were not present
when the TATA4/ML construct, in which the HIV-1 TATA box is debilitated
by point mutations (see Fig. 1A), was used as a template
(data not shown but see Fig. 7, lanes 1 and 2).
This suggests that these two classes of transcripts correspond to RNAs
correctly initiated at the HIV-1 promoter.

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Fig. 2.
An 83-85-nt doublet is produced by templates
containing a functional IST, but not by templates containing a
debilitated IST. The templates indicated above the
lanes were linearized with NcoI, which cleaves
630 nt downstream of the HIV-1 transcription initiation site, and used
to program a HeLa cell nuclear extract in the presence of radiolabeled
[ -32P]UTP. The RNA products were fractionated on a
sequencing gel. Only the top and bottom portions
of the gel, which contain the bands of interest, are shown. The bands
labeled FL correspond to full-length run-off HIV-1
transcripts, the bands labeled st correspond to short
transcripts, the bands labeled sst correspond to shorter
short transcripts derived from the template pIST/ML in which 11 base
pairs of transcribed sequences, located between the two IST
half-elements, have been deleted. The sizes of DNA markers are
indicated on the right.
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To determine whether the 83- and 85-nt long transcripts corresponded to
IST-dependent short transcripts, we transcribed several promoter constructs carrying mutations that had been characterized for
short transcript synthesis activity in vivo (3, 4). The
short transcript activity of these constructs in vivo is
summarized in Fig. 1A. In vitro, the short
transcript doublet was present with mutants ms5/ML, ms4/ML, ms3/ML, and
ms2/ML (Fig. 2, lanes 4-7), none of which exhibit selective
deficits in short transcript synthesis in vivo, but not with
mutants ms1/ML, pIST
/ML, msABC/ML, and msAB/ML, which do
not direct the formation of short transcripts in vivo
(lanes 1, 2, 9, and 10). Significantly, the
msBC/ML mutant, which directs low but detectable levels of short
transcripts in vivo (4), also directed low levels of short
transcripts in vitro (lane 11). Furthermore, the
pIST/ML construct, in which 11 base pairs of transcribed sequence
between the two IST half-elements are deleted (Fig. 1A) and
which directs the formation of correspondingly shorter short
transcripts in vivo (4), also directed the formation of
shorter short transcripts in vitro (lane 12,
bands labeled sst). The different abilities of these
constructs to direct the formation of short transcripts did not reflect
a general defect in transcription, because all constructs directed the
synthesis of significant levels of full-length transcripts (lanes
1-12, band labeled FL). Thus, the extensive correlation between the ability of the various mutants to support the
synthesis of short transcripts in vivo and the synthesis of the 83-85-nucleotide doublet in vitro suggests that
synthesis of this doublet reflects the activity of the IST element.
To allow for efficient internal labeling of the transcripts, the
cell-free transcription assay contained a much lower concentration of
UTP (7.5 µM unlabeled, 165 nM radiolabeled)
than of the other three nucleotides (250 µM each). Thus,
it was necessary to ascertain that the short transcripts did not result
from artifactual pausing or termination by the RNA polymerase due to a
rate-limiting UTP concentration. We therefore varied the concentration
of UTP in the transcription reaction from 1.0 to 250 µM
by increasing the amounts of non-radiolabeled UTP, and determined the
amounts of short transcripts and full-length transcripts generated by
the wild-type template pHIV-1/R/ML at each UTP concentration. Fig. 3A shows the results of this
experiment, and Fig. 3B the percentage of maximum amounts of
short and full-length transcripts obtained at each UTP
concentration.

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Fig. 3.
The short transcripts do not result from
artifactual pausing or termination of the polymerase at low UTP
concentrations. A, in vitro transcriptions
identical to those in Fig. 2 were performed with wild-type template
pHIV-1/R/ML in the presence of increasing concentrations of unlabeled
UTP, which are indicated above each lane (marked
[UTP]). The concentration of [ -32P]UTP
radiolabel was 330 nM (2 µl of 3000 mCi/mmol in 50 mM Tricine, pH 7.6) in all reactions. The intensity of the
bands does not reflect the number of transcripts because with
increasing unlabeled UTP concentration the ratio of unlabeled to
labeled UTP increased as indicated above the lanes (marked
[UTP]/[*UTP]). The signals corresponding to
the short and full-length HIV-1 transcripts were quantitated with a
PhosphorImager (FUJI BAS 1000) and corrected for transcript length and
uridine content, as well as for the variations in the ratio of
unlabeled to labeled UTP. The molar ratios of the 83-85 doublet to the
full-length transcripts are indicated for each UTP concentration
(marked st/FL). B, graphic representation of the
transcription depicted in A. The concentration of unlabeled
UTP is plotted along the x axis. The relative levels of
short and full-length transcripts are plotted on the y axis expressed as percentages with respect to their respective maximum levels observed at 250 µM UTP.
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As shown in Fig. 3A, the radiolabeled signal corresponding
to both short and long transcripts decreased as the ratio of unlabeled UTP to radiolabeled UTP ([UTP]/[*UTP], see top of figure)
increased, as expected. However, as shown in Fig. 3B, actual
synthesis of both the short and full-length transcripts increased
steadily as a function of total UTP concentration and approached
saturation at 250 µM UTP, as determined after
quantitation of the signals with a PhosphorImager and correction for
the increase in the unlabeled to radiolabeled UTP ratio. Remarkably,
the synthesis curves of short and full-length transcripts as a function
of UTP concentration were nearly superimposable, except for a slight
predominance of the full-length transcripts at the lowest UTP
concentrations (Fig. 3B). Indeed, the ratio of short
transcripts to full-length transcripts was nearly identical at each UTP
concentration, with the exception of the lowest concentrations of UTP,
where it was slightly lower (see of st/FL, top of Fig.
3A: compare lane 13 to lanes 1-3). If
limiting UTP concentration contributed significantly to short transcript formation, the opposite would be expected, i.e.
the ratio of short to long transcripts would be higher at the lowest concentrations of UTP. Thus, in this assay, short transcript formation does not result from RNA polymerase pausing or termination due to low
UTP concentrations. This is consistent with the observation that the
short transcripts are not observed when the various IST
mutants are transcribed, which should be equally susceptible to such
artifactual pausing or termination.
Wild-type Short Transcripts and Short Transcripts Carrying the ABC
Mutation Are Equally Stable in Vitro--
The ABC mutation changes
sequences downstream of the transcription start site that are part of
the short transcripts. These sequence changes maintain the predicted
secondary structure of the short transcripts. Moreover, in nuclear
run-on assays in vivo, which measure the rate of RNA
synthesis during a time period believed to be too short to allow for
significant RNA turnover, the ABC mutant does not support the
accumulation of short transcripts, suggesting that the defect is caused
by reduced transcription rather than by a decreased stability of the
short transcripts (4). Nonetheless, the possibility remained that,
in vitro, we failed to detect short transcripts derived from
the msABC/ML construct because of decreased stability. Therefore, we
compared the relative stabilities of wild-type short transcripts and
short transcripts carrying the ABC mutation in the in vitro
transcription reaction. Internally [
-32P]CTP-labeled
short transcripts extending from +1 to +68 were synthesized in
vitro with bacteriophage T7 RNA polymerase (see "Experimental
Procedures"). Equal amounts of either wild-type or ABC short
transcripts were then added to reactions identical to the cell-free
transcription assay except for the absence of the
[
-32P]UTP label. One-microliter aliquots were taken at
various time points and resolved on a denaturing polyacrylamide gel.
The bands corresponding to the short transcripts were then quantitated
with a PhosphorImager and the signals were plotted as depicted in Fig. 4. Both wild-type and ABC short
transcripts were degraded in the reactions with nearly identical
half-lives of approximately 80 min. This suggests that the lack of
short transcript accumulation observed with the msABC/ML construct
results indeed from a transcription defect rather than from increased
RNA turn-over. Consistent with this observation, the addition of
RNasin, an inhibitor of common eukaryotic ribonucleases (10), had no
effect on the accumulation of the short transcripts in the in
vitro transcription assay (data not shown).

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Fig. 4.
Wild-type short transcripts and short
transcripts carrying the ABC mutation have similar stabilities in the
in vitro transcription system. Wild-type short
transcripts and short transcripts with the ABC mutation were
synthesized with bacteriophage T7 RNA polymerase and incubated in
reactions identical to the in vitro transcription except
that the UTP was not radiolabeled. At the time points indicated,
aliquots were withdrawn from the reactions and the RNAs fractionated on
a sequencing gel. The bands corresponding to the short transcripts were
then quantitated with a Fuji BAS1000 PhosphorImager. The counts
present at time 0 were set at 100%.
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IST-directed Promoter-proximal Transcription Represents a
Significant Fraction of Total in Vitro Transcription--
High levels
of background bands around the 65-nt marker made it difficult to
interpret the lower part of the gels used to resolve the in
vitro transcripts. To determine whether additional IST-dependent short transcripts might be located in this
region of the gel, and to map the short and long transcripts more
accurately, we analyzed the products of an in vitro
transcription reaction by an RNase protection assay with unlabeled
antisense riboprobes (see "Experimental Procedures"). As
illustrated in Fig. 5A, in a
run-off assay the pHIV-1/R/ML and the msABC/ML constructs gave the
expected full-length transcripts (lanes 5 and 6).
As determined by quantitation of the bands with a PhosphorImager, the
amounts of full-length transcripts synthesized by the msABC/ML mutant represented about 60% the amounts of full-length transcripts
synthesized by the wild-type construct. A similar moderate reduction of
full-length transcripts is observed with this mutant in
vivo, presumably because, in addition to inactivating the DNA
sequences required for activation of short transcript synthesis, the
ABC mutations also compromise sequences that contribute to basal
transcription (4). As expected, the pHIV-1/R/ML wild-type construct,
but not the msABC/ML construct, which contains a debilitated IST,
produced the short transcript doublet (lanes 5 and
6, bands labeled st). In addition, the
pHIV-1/R/ML construct gave stronger signals in the 65-nt range than the
msABC/ML construct, suggesting that some of these high mobility bands
may represent IST-dependent short transcripts.

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Fig. 5.
IST-dependent transcription
represents a significant fraction of the total transcription from the
HIV-1 promoter in vitro. A, the templates
indicated above the lanes were linearized with NcoI and used to program a HeLa cell nuclear extract in the
presence of radiolabeled [ -32P]UTP. The transcription
reactions were identical to those described in Fig. 2, except that
poly(dG-dC)·poly(dG-dC) was added to a concentration of 750 ng/µl, and poly(I)·poly(C) to
175 ng/µl. In lanes 2 and 4, 125 ng of Tat
(recombinant protein purified from E. coli, generously
provided by M. Laspia, Dartmouth School of Medicine) were added to the
reactions. In lanes 1-4, the resulting RNAs were then
hybridized to an excess of unlabeled riboprobe hybridizing to the 146
to +329 HIV-1 region, single-stranded RNA was digested with RNase T1,
and the protected RNA fragments were resolved on a sequencing gel. In
lanes 5 and 6, the radiolabeled RNAs produced
in vitro were directly fractionated on the same sequencing
gel. In lanes 5 and 6, the bands labeled
FL have the expected length for full-length run off RNAs
initiated at the HIV-1 promoter. In lanes 1-4, the bands
labeled FL have the expected length for transcripts
protected from position +1 to position +329, where the complementarity
to the antisense riboprobe ends. The bands labeled st
correspond to short transcripts. The sizes of the DNA markers are
indicated on the right. B, quantitative analysis of
promoter-proximal and full-length transcription detected in
A. The bands corresponding to full-length transcripts or to transcripts shorter than 95 nt in lanes 1 and 3 of panel A were quantitated with a Fuji BAS1000
PhosphorImager. The numbers were normalized with respect to
the number of U residues present in each type of RNA molecules. In
addition, we corrected the short transcript signal obtained for
lane 3 for the negative effect of the ABC mutations on
general transcription by multiplying it by the ratio of full-length
transcripts observed with the wild-type construct (lane 1)
versus full-length transcripts obtained with the IST mutant
construct (lane 3). The amount of transcripts shorter than
95 nt obtained with the wild-type IST construct (lane 1 of A) was then set at 100%. The relative amount of full-length
transcripts (329 nt in length) obtained with the same construct is
shown, as well as the approximate contribution of the activity of IST to promoter-proximal transcription (cross-hatched portion of
the bar).
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We then analyzed the products of a similar in vitro
transcription reaction by RNase protection with probes derived from the pHIV-1/ML/T3/
Eco and pABC/ML/T3/
Eco constructs. The
pHIV-1/ML/T3/
Eco construct as well as the location of the probes,
which extend from positions
146 to +329 relative to the HIV-1
transcription start site, and the protected full-length and short
transcripts, are depicted in Fig. 1B. With both templates,
we observed protected RNAs of approximately 329 nt, the protected
length expected for full-length transcripts (Fig. 5A, lanes
1 and 3, band labeled FL). In addition, the
wild-type pHIV-1/R/ML template, but not the IST mutant msABC/ML
template, gave rise to a protected doublet strikingly similar to the
doublet observed in the run-off assay (compare lanes 1 and
5). Significantly, the wild-type template also gave rise to
a number of bands in the 88-95- and 50-65-nt range that were not
present with the IST mutant msABC/ML template (compare lanes
1 and 3). Thus, these transcripts, too, depend for
their synthesis on an intact IST element, and part of the signal in the
50-65-nt range observed in the run-off assay (lane 5)
actually corresponds to IST-dependent short transcripts
partially obscured by background bands.
As shown in Fig. 5B, PhosphorImager quantitation of the
pHIV-1/R/ML signals corresponding to the 329-nt full-length transcripts and those corresponding to transcripts shorter than 95 nt (see Fig.
5A, lane 1) revealed that the ratio of short to full-length RNA molecules is approximately 15:1. Thus, consistent with previous observations (11), there is a strong polarity of transcriptional activity from promoter-proximal to promoter distal locations. In
addition, comparison of the amounts of short transcripts obtained with
the wild-type IST construct pHIV-1/R/ML and the mutated IST construct
msABC/ML reveals that, after correction for the effect of the ABC
mutations on general transcription and for the additional (radiolabeled) U residues present in the mutated RNAs (see Fig. 1A), the activity of IST accounts for approximately 70-80%
of the promoter-proximal transcription detectable in this assay (Fig. 5B). Together, these results suggest that under these
conditions IST-directed transcription represents a significant portion
of the total in vitro transcription derived from the HIV-1
promoter.
It has been shown previously that the HIV-1 transcriptional activator
Tat increases the levels of full-length transcripts but reduces the
levels of the IST-dependent short transcripts (2, 4). We
therefore tested whether addition of Tat to the in vitro
transcription reactions would affect the levels of short transcripts in
this manner. As shown in Fig. 5A, under our transcription conditions optimized for short transcript synthesis, addition of Tat
trans-activated the full-length transcripts only poorly (compare lane 2 to lane 1 and lane 4 to lane 3, band labeled FL). However, consistent
with the in vivo observations, the weak bands in the
88-95-nt range, the 83-85-nt short transcript doublet, and the
shorter transcripts were reduced in the presence of Tat (compare
lanes 2 and 1). Thus, the in vitro
assay faithfully reproduces this important in vivo
characteristic of short transcripts, further suggesting that the short
transcripts detected in vitro represent IST activity.
Because the properties of the 83-85-nucleotide doublet were
representative of the total population of short transcripts revealed in
the RNase protection assay, IST activity in vitro was
henceforth assayed by accumulation of this doublet in the simpler
run-off assay.
The Short Transcripts Are Synthesized by RNA Polymerase
II--
Short, nonpolyadenylated RNAs are frequently synthesized by
RNA polymerase III. Thus, an intriguing possibility was that the HIV-1
short transcripts were also synthesized by this RNA polymerase. To
address this question, we determined the sensitivity of short and long
transcript synthesis to various concentrations of the fungal toxin
-amanitin. As depicted in Fig.
6A, synthesis of both the
full-length and short HIV-1 transcripts was sensitive to the low
concentrations of
-amanitin that typically inhibit RNA polymerase II
transcription (lanes 2-5). In contrast, the synthesis of
VAI RNA, an RNA polymerase III transcript, was inhibited only at the
high concentrations of
-amanitin that typically inhibit RNA
polymerase III transcription (Fig. 6B). Thus, like the
full-length transcripts, the HIV-1 short transcripts are synthesized by
RNA polymerase II in vitro.

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Fig. 6.
The short transcripts are synthesized by RNA
polymerase II. A, the HIV-1 templates indicated
above the lanes were linearized with NcoI and
used to program a HeLa cell nuclear extract in the presence of
radiolabeled [ -32P]UTP and the concentrations of
-amanitin indicated above the lanes. Only the
top and bottom portions of the gel, which contain the bands of interest, are shown. The bands are labeled as in Fig. 1.
B, the reactions were performed as in A, except
that the Ad2 VAI gene, an RNA polymerase III gene, was used as
template. The band labeled VAI corresponds to correctly
initiated VAI RNA.
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Synthesis of the Short but Not the Full-length Transcripts Is
Resistant to DRB--
The purine nucleoside analog DRB inhibits the
elongation phase of RNA polymerase II transcription in vitro
(12). Since IST appears to direct the formation of transcription
complexes incapable of efficient transcription elongation, an
intriguing possibility is that the synthesis of the short transcripts
is DRB-resistant. To address this question, in vitro
transcription reactions were performed with the wild-type and two IST
mutant templates in the absence or presence of DRB at a concentration
that has been shown to inhibit formation of HIV-1 full-length
transcripts in vitro (11). As expected, DRB abolished the
synthesis of the 630-nucleotide run-off transcript (Fig.
7, compare lanes 2-4 and
6-8, bands labeled FL). In contrast, it had no
effect on the synthesis of the IST-dependent short
transcripts (compare lanes 2-4 and 6-8, bands
labeled st). This observation indicates that the synthesis
of the full-length transcripts is not a prerequisite for the synthesis
of the short transcripts, and thus confirms that the short transcripts
do not arise from degradation or processing of long transcripts. In
addition, the amounts of short transcripts were not increased in the
presence of DRB, and no short transcripts were induced when
IST
mutants msABC/ML and IST-/ML were transcribed in the
presence of DRB (compare lanes 7 and 8 to
lane 6; see also Fig.
9B, lanes 5 and 7).
Therefore, DRB does not appear to convert elongation competent
transcription complexes into elongation incompetent ones. It rather
seems to inhibit the assembly of elongation competent transcription
complexes at the promoter. And most importantly, this observation
supports a model in which short and full-length transcripts are the
products of two types of transcription complexes with distinct
biochemical properties: unlike formation of the transcription complex
giving rise to full-length transcripts, formation of the transcription
complex giving rise to the short transcripts does not require the
activity of factor(s) inhibited by DRB.

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Fig. 7.
The synthesis of short, but not full-length,
transcripts is resistant to DRB. The templates indicated
above the lanes were linearized with NcoI and
used to program a HeLa cell nuclear extract either in the absence
(lanes 1-4) or presence (lanes 5-8) of 160 µM DRB. The bands are labeled as in Fig. 2. The location of DNA markers is indicated on the right. Only the
top and bottom portions of the gel, which contain
the bands of interest, are shown.
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Fig. 8.
Varying several transcription reaction
parameters affects synthesis of short and full-length transcripts
differentially. A, the pHIV-1/R/ML template was linearized
with NcoI and used to program a HeLa cell nuclear extract.
The reactions contained the indicated amounts of poly(I)·poly(C). The
bands corresponding to full-length and short transcripts were
quantitated with a Fuji BAS1000 PhosphorImager, and the counts present
with no added poly(I)·poly(C) were set at 100%. B, the
reactions were performed and analyzed as in A except that
the reactions contained the indicated amounts of
poly(dG-dC)·poly(dG-dC) instead of poly(I)·poly(C). C,
the transcription was performed with the templates indicated
above the lanes, and the MgCl2 concentration was
varied as follows: lanes 1-3 contained 2.5 mM
MgCl2 derived from the nuclear extract. In lanes
4-10, additional MgCl2 was provided to the following final concentrations: 3.75 mM (lane 4), 5.0 mM (lane 5), 6.25 mM (lane
6), 7.5 mM (lane 7), 8.75 mM
(lane 8), 10.0 mM (lane 9), 11.25 mM (lane 10). Only the top and
bottom portions of the gel, which contain the bands of
interest, are shown.
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Differential Reaction Requirements for Full-length and Short
Transcript Synthesis--
While optimizing conditions for the in
vitro transcription reactions, we noticed that various reaction
parameters affected the synthesis of short and long transcripts
differentially. For example, Fig. 8, A and B,
show that increasing amounts of either poly(I)·poly(C) or
poly(dG-dC)·poly(dG-dC) inhibited full-length transcription but
stimulated the synthesis of the short transcripts over a broad
concentration range. Similarly, synthesis of short transcripts had a
much sharper MgCl2 optimum than that of the full-length
transcripts (Fig. 8C, lanes 3-10, compare FL and
st bands). No short transcripts were induced by varying the
concentrations of the above nucleic acid competitors or
MgCl2 when the mutant IST construct msABC/ML was
transcribed (data not shown).
Fig. 9A shows the effects of
adding increasing amounts of NaCl to the transcription reaction. When
the salt concentration in the reaction was raised by adding small
increments of NaCl in addition to the 50 mM KCl contributed
to the reactions by the nuclear extract, synthesis of the full-length
transcripts decreased steadily (lanes 2-7). In contrast,
the synthesis of short transcripts was first stimulated to a maximum at
80 mM salt (30 mM NaCl added, lane
4) and was inhibited only at higher salt concentrations
(lanes 5-7). To rule out the possibility that the
stimulatory effect of increased NaCl on short transcript synthesis was
due to a conversion of full-length transcripts into short transcripts,
for example, by inhibition of transcript elongation or induction of an
RNA processing activity, we wished to test whether addition of NaCl to
the reaction would modulate short transcript synthesis in the absence
of the full-length transcripts. In vitro transcriptions were
performed in the presence of different salt concentrations and the
nucleoside analog DRB, which, as shown above (Fig. 7), selectively
inhibits formation of the full-length transcripts. As expected, in the
absence of DRB, raising the total salt concentration to 80 mM by adding 30 mM NaCl to the reaction
severely reduced the level of the full-length transcripts and,
conversely, stimulated the level of the short transcripts (Fig.
9B, compare lanes 1 and 2).
Significantly, addition of NaCl stimulated short transcript synthesis
to a similar degree when the full-length transcripts were absent due to
the addition of DRB (lanes 3 and 4). The addition of NaCl did not lead to the appearance of short transcripts when the
IST
template msABC/ML was transcribed in an identical
experiment (compare lanes 5 and 6, and
7 and 8). Thus, the increase in short transcripts
observed at increased NaCl concentrations, and probably also that
observed at certain MgCl2 and nucleic acid competitor concentrations, most likely results from a stimulation of IST activity
and not from a general inhibitory effect on transcription elongation or
modulation of an RNA processing activity.

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Fig. 9.
A, the transcriptions were performed
with the templates indicated above the lanes. All reactions
contained 50 mM KCl from the nuclear extract. In
lanes 3-7, NaCl was added to the following concentrations:
15 mM (lane 3), 30 mM (lane
4), 50 mM (lane 5), 75 mM
(lane 6), 100 mM (lane 7). Only the
top and bottom portions of the gel, which contain
the bands of interest, are shown. B, the transcriptions were
performed with the templates indicated above the lanes. All
reactions contained 50 mM KCl from the nuclear extract. DRB
was added to 160 µM in lanes 3, 4, 7, and
8. NaCl was added to 30 mM in lanes 2, 4, 6, and 8, resulting in a total salt concentration of 80 mM in those lanes.
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Together, these results demonstrate that in vitro, as
in vivo (3, 4), the short and the full-length transcripts
arise from separable processes that can be modulated independently. They lend further support to the notion that short and full-length transcripts are synthesized by different transcription complexes with
distinct biochemical properties.
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DISCUSSION |
The Short HIV-1 Transcripts Observed in Vitro Result from
IST-dependent RNA Polymerase II Transcription--
Several
lines of evidence suggest that the short transcripts observed in the
in vitro transcription system reflect the activity of the
IST element that we have previously characterized in vivo. First, mutations that debilitate the IST element in vivo
severely reduce the accumulation of short transcripts in
vitro. In the case of the msABC mutation, we have shown that this
is not due to decreased stability of short transcripts carrying the ABC
mutation, as these transcripts are degraded at the same rate as
wild-type short transcripts. Second, as in vivo, the Tat
protein decreases the level of short transcripts. Third, since the
ratios of short to full-length transcripts are similar throughout a
broad UTP concentration spectrum, the short transcripts do not arise
from artifactual polymerase pausing or termination due to the low UTP concentration in our assay. Finally, by selectively abolishing synthesis of the full-length transcripts but not the short transcripts with DRB or NaCl, we have also shown that the short transcripts are not
breakdown products of the full-length transcripts.
There is, however, a discrepancy. Whereas in vivo, the RNase
T1 protection assay reveals short transcripts protected over 58, 62, and 65 nt, RNase T1 protection of the in vitro transcripts reveals a series of molecules in the 50-65-nt range and a doublet of
about 83-85 nt. These differences are likely to reflect, at least in
part, differences in stability of short transcripts of different
lengths in vitro and in vivo. Perhaps the very
short RNAs detected in vitro are not stable in
vivo because they are too short to fold into the TAR stem-and-loop
structure and are thus degraded. Similarly, the longer RNAs detected
in vitro may not be stable in vivo because they
are processed to the base of the stem-and-loop structure. Indeed,
pulse-chase studies with isolated nuclei have shown that heterogeneous,
longer precursors to HIV-1 short transcripts exist in vivo,
which are then shortened by a RNA processing activity (13). Thus,
although the sizes of the short transcripts detected in vivo
and in vitro differ, we have established a cell-free
transcription assay that faithfully reproduces the transcriptional
activity of the HIV-1 IST element, and it will now be possible to study
the biochemical events underlying its function.
Several RNA polymerase II promoters can also direct RNA polymerase III
transcription under certain circumstances. For instance, the c-Myc
promoter can support the synthesis of short, RNA polymerase III
transcripts both when injected into Xenopus oocytes (14), and in vitro (15). The human T-lymphotropic virus type 1 promoter can direct the synthesis of RNAs that are initiated at the
same nucleotide as the RNA polymerase II transcripts but are
synthesized by RNA polymerase III, apparently recruited to the promoter
in part by RNA polymerase II transcription factors (16). Because the
HIV-1 short transcripts are stable in vivo, it has been
difficult to determine their sensitivity to
-amanitin in
transfection experiments. Here, we show that, as determined by their
sensitivity to low concentrations of
-amanitin, the IST-directed
HIV-1 short transcripts synthesized in vitro are the
products of RNA polymerase II. This suggests that in vivo,
too, IST-directed transcription is mediated by RNA polymerase II.
The formation of short HIV-1 and HIV-2 transcripts in vitro
was first reported by Toohey and Jones (17). In their system, efficient
formation of short transcripts required the addition of Sarkosyl to the
transcription assay. However, the observations that Sarkosyl induced a
processing activity in the nuclear extract, and that the short
transcripts appeared after the full-length transcripts, suggested that
the short transcripts detected in that system resulted from processing
of the full-length transcripts (17). It seems unlikely, therefore, that
the short transcripts observed in that system reflected activity of the
IST element. In contrast, in their in vitro studies of Tat
trans-activation, Laspia et al. (7) detected
short transcripts of 80-85 nucleotides in length whose synthesis
decreased upon the addition of Tat to the reaction. Even though we did
not observe the same response of short transcript synthesis to some
reaction parameters described by Laspia et al. (7)
(e.g. stimulation by template preincubation, presynthesis in
the absence of radiolabeled nucleotides, or addition of Sarkosyl, data
not shown), it is possible that their short transcripts are related to
the 83-85-nucleotide doublet detected in our assay. It also seems
likely that the steep gradient of transcription downstream of the HIV-1
promoter observed previously in vitro (11) reflects in large
part the activity of the IST element. Indeed, in these experiments, the
addition of DRB inhibited Tat-activated transcription but had little
effect on promoter-proximal transcription.
Two Types of Transcription Complexes Are Assembled at the HIV-1
Promoter--
Our in vivo studies of the IST element have
suggested that the HIV-1 promoter directs the assembly of two types of
transcription complexes that differ in their elongation properties (2,
4): one type, whose formation is stimulated by Tat binding to the TAR
element, is elongation competent, while the second type, whose formation is stimulated by IST, is incapable of efficient elongation. Although these two types of transcription complexes do not appear to
differ in their use of the TATA-box binding protein (5), they probably
contain different factors. Thus, the IST element, which is not required
for synthesis of the full-length transcripts, is likely to correspond
to the binding site for a factor that specifically stimulates the
assembly of such elongation-incompetent complexes. Indeed, we have
recently identified and purified a factor, FBI-1 (factor
that binds to IST), whose binding to wild-type IST and various IST mutants correlates extensively with the abilities of these ISTs to direct the synthesis of short transcripts in vivo (6).
In addition, our observation that in vitro, the synthesis of
short and long transcripts is differentially affected by variations in
NaCl, MgCl2, DRB, and nonspecific nucleic acid
concentrations indicates that they are directed by transcription
complexes with different biochemical properties. Most striking is the
differential effect of DRB, which abolishes the synthesis of the
full-length transcripts but has no effect on that of the short
transcripts. DRB has been shown to inhibit various cellular protein
kinases including the TFIIH-associated cyclin-dependent
kinase (18), the Drosophila-positive transcription
elongation factor p-TEFb (19), and a Tat-associated kinase referred to
as TAK (20, 21), which most likely corresponds to the human homolog of
p-TEFb (22, 23). The TFIIH-associated cyclin dependent kinase, p-TEFb, and TAK can all associate with Tat (22-24) and phosphorylate the carboxyl-terminal domain of RNA polymerase II (18, 25), an event that
is thought to be required for the transition into the elongation mode
of RNA polymerase II. These observations, together with the finding
that Tat trans-activation, but not the formation of short
transcripts in the absence of Tat, requires the RNA polymerase II
carboxyl-terminal domain (21, 26), is consistent with a model in which
the IST-directed transcription complexes do not depend on certain
carboxyl-terminal domain kinases and are, therefore, resistant to DRB.
In these IST-directed transcription complexes, the RNA polymerase II
carboxyl-terminal domain would remain largely unphosphorylated,
resulting in poor elongation properties. Alternatively, or in
addition, the IST element may serve as an entry point for a
transcription complex prone to premature transcription
termination.
We thank M. Laspia (Dartmouth School of
Medicine) for helpful discussion and for providing Tat protein and
plasmid pU3RIII/pUC119, B. Ma for help with nuclear extract preparation
and DNA sequencing, R. Ratnasabapathy for plasmid pET7/R, S. Pendergrast for valuable discussion and comments on the manuscript,
M. Ockler, J. Duffy, and P. Renna for artwork and photography, V. Mittal for expert assistance with graphics, and T. Kuhlman and E. Ford
for support.