(Received for publication, August 19, 1994; and in revised form, November 1, 1994)
From the
The cellular factor, LBP-1, can repress HIV-1 transcription by preventing the binding of TFIID to the promoter. Here we have analyzed the effect of recombinant LBP-1 on HIV-1 transcription in vitro by using a ``pulse-chase'' assay. LBP-1 had no effect on initiation from a preformed preinitiation complex and elongation to position +13 (``pulse''). However, addition of LBP-1 after RNA polymerase was stalled at +13 strongly inhibited further elongation (``chase'') by reducing RNA polymerase processivity. Severe mutations of the high affinity LBP-1 binding sites between -4 and +21 did not relieve the LBP-1-dependent block. However, LBP-1 could bind independently to upstream low affinity sites (-80 to -4), suggesting that these sites mediate the effect of LBP-1 on elongation. These results demonstrate a novel function of LBP-1, restricting HIV-1 transcription at the level of elongation. In addition, Tat was found to suppress the antiprocessivity effect of LBP-1 on HIV-1 transcription in nuclear extracts. These findings strongly suggest that LBP-1 may provide a natural mechanism for restricting the elongation of HIV-1 transcripts and that this may be a target for the action of Tat in enhancing transcription.
Transcription from the HIV-1 ()promoter is regulated
through upstream elements (-120 to -77) by the inducible
activator NF-
B and through a downstream element (+14 to
+44) by the virus-coded Tat protein (reviewed in (1) ). In
between lie DNA elements that are recognized by the constitutive
activator SP1, which may facilitate Tat and NF-
B
functions(2, 3) , and core promoter elements (TATA and
Initiator) which are recognized by the basic transcriptional
machinery(4, 5, 6) . Embedded within the
latter elements are additional sites, recognized minimally by the
cellular factor LBP-1 (7, 8) , that may contribute to
basal promoter activity.
The viral Tat protein is essential for
virus replication and activates transcription throughout a stem-loop
structure (TAR) in the nascent HIV-1 mRNA(1) , most likely in
conjunction with other cellular cofactors (9, 10 and references
therein). Various in vivo studies have documented stimulatory
effects of Tat on transcriptional elongation (11, 12, 13, 14) and, in some cases,
on transcription initiation as
well(12, 13, 14, 15) . Consistent
with a more commonly observed effect of Tat on elongation in
vivo, in vitro studies have revealed a large effect of
Tat mainly at the level of elongation and further documented distinct
classes (weakly versus highly processive) of elongation
complexes(16, 17, 18, 19, 20) .
While the precise mechanism for Tat effects on elongation is unknown,
the simplest model invokes an alteration in processivity of the
elongation complex in a co-transcriptional manner, possibly analogous
to the action of the RNA-bound N protein in conferring
anti-termination properties on the associated RNA
polymerase(21) . On the other hand, and consistent with
possible effects of Tat on levels of initiation as well, it has been
shown that activation by Tat (but not NF-
B) requires a specific
TATA element(3, 22, 23) , that Tat can
function when tethered to the promoter by fused DNA binding
domains(24, 25, 26, 27) , and that
Tat can stabilize preinitiation complexes(20) . To explain
these diverse phenomena, it has been suggested (12, 13, 28, 29, 30) that
TAR-bound Tat could function during assembly of the subsequent PIC
(``reach back'' mechanism) to effect formation of a more
processive complex, analogous to that formed by conventional
activators, and possibly the rate of initiation as well.
Possibly related to the issue of Tat function is the demonstration of short stable HIV-1 transcripts that may either decrease or remain unchanged (depending on the assay) following induction by Tat of long transcripts(11, 31, 32, 33, 34, 35) . While such transcripts could be derived (possibly by processing; (30) and (33) ) from less processive elongation complexes, their formation could provide initial RNA binding sites for Tat function. A downstream element (-1 to +26), designated IST, that induces synthesis of short transcripts has been described (32, 35) . While IST mutations eliminating short transcript synthesis failed to eliminate Tat induction of long transcripts(35) , it has been suggested (28) that the low but demonstrable basal transcription in the transfection assays employed might generate sufficient nascent RNA for initiation of the Tat response. The cellular factors responsible for IST function are unknown. However, the cellular factor LBP-1 was previously shown (7, 8, 37) to interact in a concentration-dependent manner with a high affinity site (-4 to +21) overlapping both Inr-like elements (5, 6) and the IST element (32, 35) and with a low affinity site (-38 to -16) that overlaps the TATA box. Promoter mutagenesis studies led to the conclusion that LBP-1 is not involved in either IST function (35) or Tat induction(36) , but studies to be described here leave this possibility open. While LBP-1 per se has not been shown to activate transcription via the high affinity recognition sites which contribute to promoter activity(7, 37) , high concentrations of this factor have been shown to repress transcription initiation when bound to upstream sites prior to TFIID binding to the interspersed TATA element(37) . Here, we document the ability of a recombinant LBP-1 isoform (38) to reduce the processivity of RNA polymerase initiated at the HIV-1 promoter, and we discuss a possible relationship to Tat-induced transcription.
Figure 5: Analysis of the LBP-1b elongation block on normal and mutated (IS4) promoters. PICs formed with both HIV-1 (WT DNA) and HIV-1/IS4 (IS4 DNA) promoters were isolated as described under ``Experimental Procedures'' and analyzed by the pulse-chase assay. After the pulse, increasing amounts of LBP-1b were added as indicated (lanes 3, 4, 7, and 8), and reaction mixtures were incubated at 30 °C for 7 min. The chase was performed thereafter for 20 min at 30 °C. The reaction products from the pulse are indicated as 13-mer (lanes 1 and 5) and from the chase as 600 nt (lanes 2 and 6). However, because of base substitution near the start site in IS4 (below), the corresponding paused transcripts are several nucleotides shorter. A schematic drawing in the bottom of the figure shows the HIV-1 promoter containing low affinity LBP-1 binding sites flanking the TATA box and either wild type or mutated (IS4) high affinity LBP-1 recognition sites. The DNA sequence of the Wt promoter region from -4 to +21 is ACTGGGTCTCTCTGGTTAGACCAGA (LBP-1 binding sites underlined) while that of the Mt/IS4 promoter on this region is AGATGGTCTCTAGTGTTAGCAAAGA (mutated residues in bold type)(37) .
Figure 8:
Effect of Tat on elongation block of HIV-1
transcripts by LBP-1b. Standard transcription assays were performed, as
indicated in the figure and detailed under ``Experimental
Procedures,'' with 50 ng each of HIV-1 and AdML promoters and 100
ng of poly(dI-dC). PIC formation was followed by the addition of LBP-1,
Tat, GTP, CTP, ATP, and [-
P]UTP and further
incubation for 60 min at 30 °C.
Figure 1:
Specific inhibition of
HIV-1 transcription by recombinant LBP-1b in vitro. The
pulse-chase analysis was performed with unfractionated nuclear extract
as indicated in the figure and detailed under ``Experimental
Procedures.'' After PIC formation, transcription from both HIV-1
and AdML promoters was initiated with the addition of dATP, GTP, CTP
and [-
P]UTP (pulse) in the presence or
absence of LBP-1 as indicated.
All reactions were terminated by addition of 50 µl of 25 mM EDTA solution and extracted with phenol/chloroform. Nucleic acids were precipitated with ethanol after addition of yeast tRNA and ammonium acetate to final concentrations of 20 µg/ml and 3 M, pH 7.5, respectively. Reaction products ( Fig. 1and Fig. 8) were analyzed by electrophoresis through a denaturing 6% polyacrylamide gel containing 50% urea. In order to determine the level of either activation or repression of HIV-1 transcription by Tat or LBP-1, respectively, labeled bands were excised from the gel, and the radioactivity was measured by liquid scintillation counting.
Figure 2: Kinetic analysis of HIV-1 transcript formation in a single round of transcription. The pulse-chase assay with isolated PICs was performed as indicated in the figure and detailed under ``Experimental Procedures,'' except that the KCl concentration was 50 mM. The transcription products from the pulse are shown in lane 1 (indicated as 13-mer), and those from the chase are shown in lanes 2-8 (full-length run-off transcripts are 600 nt). Numbers on the top of each lane indicate the period of time for pulse (lane 1) and chase (lanes 2-8), respectively. In lane 8, after a 10-min chase, 200 mM KCl (*) was added to the reaction and further incubated at 30 °C for 10 min. The arrow shows a short RNA transcript of 70 nt suggesting that RNA polymerase pauses at position +70 downstream of the promoter. The other discrete pause site evident in lanes 2-8 lies near position +110. It should be noted that in this kind of assay the amount of radioactivity present in any given lane does not change by more than 10% (C. A. Parada, J.-B. Yoon, and R. G. Roeder, unpublished observation). Furthermore, in other analyses, the distribution of transcripts observed after a 10-min chase (lane 7) remained unchanged even after a 60-min chase (data not shown).
Figure 3: Effect of recombinant LBP-1b on HIV-1 transcription in vitro. Pulse-chase assays were performed with isolated PICs as diagrammed in Fig. 2and detailed under ``Experimental Procedures.'' A, lack of an effect of LBP-1b on initiation/early elongation of HIV-1 transcripts. HIV-1 PICs were isolated under standard transcription reaction conditions (without NTPs) at 70 mM KCl. PICs were then incubated either in the absence (lane 1) or in the presence of increasing amounts of LBP-1 (lanes 2 and 3) for 7 min at 30 °C before performing a pulse (P) reaction as in Fig. 2. The various transcription reaction products are indicated. B, block of elongation of HIV-1 transcripts by LBP-1b. After the formation of an early elongation complex with RNA polymerase stalled at position +13 (pulse), as detailed under ``Experimental Procedures,'' 50 ng and 100 ng of LBP-1b were added to transcription reactions in the absence (lanes 1-4) or presence (lanes 5-8) of 60 µg of HeLa nuclear extract and incubated for 7 min at 30 °C. Unlabeled NTPs were then added, and the reaction mixture was further incubated for another 20 min to score formation of 600-nt transcripts (chase). Lanes 1 and 5 show the reaction products (13-mer) of the pulse (P). Numbers on the side refer to transcript size markers.
Figure 4: Characterization of the HIV-1 transcription block by LBP-1b. A, effect of KCl on HIV-1 elongation complexes paused by LBP-1b. Pulse-chase assays were performed with isolated PICs either in the absence (lanes 1 and 3) or in the presence (lanes 2 and 4) of LBP-1b as diagrammed in Fig. 2and detailed in the legend to Fig. 3. In addition, 200 mM KCl was added to the reaction in lane 4 (*) after the standard chase, and all reactions then were incubated for an additional 20 min at 30 °C. The paused transcripts observed after a 20-min chase under standard conditions (lane 3) were not further extended even in a 60-min chase. B, reversal of LBP-1b elongation block by oligonucleotides that bind LBP-1. The pulse was performed as diagrammed in Fig. 2and detailed under ``Experimental Procedures.'' LBP-1b (100 ng) was then added either alone (lane 3) or with an oligonucleotide containing high affinity LBP-1 recognition sites (Wt, lane 4), or with an oligonucleotide containing mutations in high affinity LBP-1 recognition sites (IS4 mutation, see Fig. 5legend) (Mt, lane 5). The reaction mixtures were then incubated for 7 min at 30 °C prior to execution of the chase. Both Wt and Mt oligonucleotides were present at a 100-fold molar excess over the DNA template. Lanes 1 and 2 show the products of control pulse (13-mer) and chase (600 nt) reactions, respectively.
During the course of our studies on repression of HIV-1 transcription initiation by LBP-1b in extracts from HeLa cells, it was observed that recombinant LBP-1b (hereafter referred to as LBP-1 in this paper) could inhibit transcription in a dose-dependent manner even when added after preinitiation complex (PIC) formation. Thus, in the analysis of Fig. 1, the HIV-1 template and a control adenovirus MLP template were preincubated with nuclear extract prior to addition of LBP-1. Transcription was subsequently scored by the pulse-chase assay diagrammed in Fig. 1(see below also). Since these assay conditions preclude the inhibition of TFIID binding to the promoter by exogenous LBP-1 (by preforming a stable PIC) and score only a single round of transcription(37) , it was surprising to find that even under these conditions LBP-1 specifically inhibited HIV-1 transcription and not MLP transcription (Fig. 1, lanes 1-4). This result suggested that LBP-1 could also repress HIV-1 transcription by a mechanism different from that involving exclusion of TFIID binding to the TATA box. Moreover, this effect was specific for the HIV-1 promoter when compared to a control adenovirus promoter (MLP) lacking LBP-1 binding sites.
The kinetic analysis during the chase showed a clear
pause site near +70, with the accumulated transcripts chased as a
function of time (Fig. 2, lanes 2-7). This pause
was observed previously both in vitro, with a
(dC)-tailed HIV-1 promoter which allows initiation
independent of promoter elements(50) , and in
vivo(11, 32, 33, 34, 35) .
Thus, it was interesting to find that, under our assay conditions, the
RNA polymerase pauses at +70 even at high concentrations of KCl
and sarkosyl where the elongation complex is highly processive (data
not shown). The mechanistic relevance of this pause to Tat function is
presented under ``Discussion.''
It is worth noting that LBP-1 binds as a dimer to its recognition site (38) and that the HIV-1 promoter contains three high affinity and six low affinity LBP-1 recognition sites (Fig. 7). Thus, the molar ratio of LBP-1 to the HIV-1 promoter (relative to the above considerations) is no more than 2 when 100 ng of LBP-1 are present in any given reaction. This indicates that a low concentration of recombinant LBP-1 in our assay conditions can lead to a block of elongation.
Figure 7:
Binding of LBP-1b to upstream low affinity
sites on the HIV-1 promoter. Gel mobility shift analysis was performed
with 5 ng of LBP-1b and 20 fmol of a radiolabeled DNA fragment
(-120 to -31) derived from the HIV-1 promoter. Competitor
oligonucleotides, used at 100-fold molar excess compared to the probe,
are indicated. Both ``long'' (L) and
``short'' (S) are double-stranded oligonucleotides
that contain three or two high affinity LBP-1 sites and correspond to
position -17 to +27 and -16 to +15, respectively.
The L oligonucleotide contains mutated high affinity sites
(IS4). The drawing below the figure shows the HIV-1 promoter as well as
the location of the probe and competitors used in this assay. Low
affinity LBP-1 binding sites that flank the TATA box (37) and
the potential low affinity LBP-1 binding sites (-68
CCTGGGCGGGACTGG -53) that overlap the Sp1 binding sites
(-77 to -45) are also
indicated.
The pulse-chase assay involves PIC purification by gel filtration which removes most (approximately 90%) of the HeLa nuclear proteins. In order to mimic more closely the conditions under which exogenous LBP-1 inhibited the HIV-1 transcription when PICs were preformed in a standard in vitro system (unfractionated nuclear extract) (Fig. 1), we studied the effect of LBP-1 in a pulse-chase assay in which nuclear extract proteins were added back (with LBP-1) during the chase (Fig. 3B, right). Under these conditions, the elongation block proximal to the promoter was overcome totally by the presence of the extract proteins with promoter distal elongation products clearly evident (Fig. 3B, compare lanes 7 and 8 with lane 6). Significantly, however, the transcripts were heterogeneous in size (mostly from 150 to 400 nt), and the level of full-length (600 nt) run-off transcripts was still decreased in an LBP-1 concentration-dependent manner. This was revealed more clearly when the transcription products were fractionated in a 6% denaturing polyacrylamide gel (data not shown). Although we do not know how HeLa nuclear extract components overcome the LBP-1 block proximal to the promoter, it is likely that this involves endogenous elongation factors such as TFIIF, TFIIS, and TFIIX (references above) or the more recently described elongation activities such as P-TEF (46) and SIII (51) in the extract. At the same time, it is significant that even in the presence of HeLa nuclear extract factors, LBP-1 can still restrict the processivity of RNA polymerase and result in a potential block of elongation distal to the promoter. This result correlates with the inhibition of HIV-1 transcription by LBP-1 in the crude cell-free system under conditions where PICs are preformed before addition of LBP-1 (Fig. 1).
Since the pulse-chase analysis at both 50 and 70 mM KCl showed a restriction of HIV-1 transcription at the level of elongation in the absence of exogenously added LBP-1 (Fig. 2, 3B, 4A, 4B, and 5), we tested whether the potential recruitment of endogenous LBP-1 in HeLa nuclear extracts into the PIC was involved in this restriction. However, even when LBP-1-depleted nuclear extracts were employed for PIC formation and isolation, HIV-1 transcription was still restricted at the level of elongation (data not shown). As mentioned above, this restriction most likely reflects the absence of elongation factors in isolated PICs(18, 46, 47, 52) . Furthermore, since the LBP-1 block of elongation is observed when LBP-1 is added either after PIC formation (Fig. 1) or after 13-mer formation (Fig. 3B), it seems likely that LBP-1 might function during early elongation events by competing with elongation factors (see ``Discussion''). We do not know why HIV-1 transcription is not restricted at the level of elongation in standard (untreated) HeLa nuclear extracts ( Fig. 1and Fig. 8). However, since the sole addition of a low concentration of recombinant LBP-1 leads to a block of elongation ( Fig. 1and Fig. 8), this argues for a model in which LBP-1 function may be subject either to a competition with elongation factors or to a tight regulation (i.e. by phosphorylation or by an interacting repressor) which may be altered during nuclear extract isolation and manipulation.
We next tested whether the DNA binding activity of LBP-1 was required for the HIV-1 promoter elongation block. Evidence for this was indicated both by the ability of oligonucleotides containing wild type, but not mutated, LBP-1 binding sites to reverse the effect of LBP-1 (Fig. 4B, compare lanes 4 and 5 with lane 3) and by the inability of LBP-1d (the isoform which does not bind to DNA) to block HIV-1 elongation (data not shown).
The failure of mutations in the high affinity LBP-1 recognition sites to relieve the inhibition of elongation by LBP-1 suggested that the binding of LBP-1 to these sites might not be the sole mechanism by which LBP-1 can block HIV-1 elongation. Thus, it remained possible that LBP-1 could still bind to the template under the conditions employed, either to the mutated high affinity LBP-1 sites themselves or, in an independent manner, to the low affinity sites located immediately upstream of high affinity sites (between -4 and -80). To gain insight into this question, a gel retardation assay was employed to test the direct binding of LBP-1 to radiolabeled oligonucleotide probes containing either wild type or mutated high affinity LBP-1 binding sites. Somewhat surprisingly, LBP-1 still bound to a probe containing the mutated LBP-1 sites, although the binding was 4-fold lower when compared with binding to the wild type sites (Fig. 6, compare lanes 9-12 with lanes 2-4, respectively). Interestingly, when poly(dI-dC) was included in the reaction as a nonspecific competitor, LBP-1 bound with a much lower efficiency (more than 40-fold) to the mutated LBP-1 probe relative to the wild type probe (Fig. 6, compare lanes 13-15 with lanes 5-7, respectively). Thus, the binding of LBP-1 to the high affinity sites compared to the low affinity sites becomes more prominent in the presence of nonspecific competitors. This observation could explain why mutations of high affinity LBP-1 recognition sites (IS4) relieved the inhibition of HIV-1 transcription at the level of initiation when poly(dI-dC) was present in the transcription reaction(37) .
Figure 6: Binding of LBP-1b to both wild type (Wt) and mutant (Mt(IS4)) high affinity LBP-1 sites. The gel mobility shift assay was performed as described under ``Experimental Procedures.'' Ten fmol of either Wt probe (lanes 1-7) or Mt (IS4) probe (lanes 8-15) was mixed with or without the indicated amounts of poly(dI-dC) before the addition of LBP-1b as indicated. Both the free and bound probe are shown by the brackets.
Competition analyses
with the gel retardation assay indicated that the binding of LBP-1 both
to the wild type and to the mutated LBP-1 sites was specific even
though the binding affinity to the latter sites was low. Furthermore,
although it is unclear what the difference is between the lower and
upper bands in the LBP-1DNA complex (Fig. 6), both
complexes are specifically bound by LBP-1 (data not shown). Hence, it
remained possible that LBP-1 could bind to mutated sites with higher
efficiency in the context of the HIV-1 promoter and other interacting
factors, even in the presence of poly(dI-dC), and thus block
elongation. To further investigate this possibility, we used the
pulse-chase assay to test for elongation blocks by LBP-1 on two HIV-1
promoters containing different and more drastic mutations in the high
affinity LBP-1 binding sites. Although DNA probes containing these
mutations did not exhibit LBP-1 binding by any (direct binding or
oligonucleotide competition) assays, templates with these mutations
showed the same sensitivity to elongation inhibition by LBP-1 as did
templates with HIV-l/IS4 and wild type promoter (data not shown). Taken
together, these results indicate that the binding of LBP-1 to high
affinity sites is not necessary for LBP-1 to restrict elongation of
HIV-1 transcripts.
Thus, it is evident that LBP-1 can bind independently (in the absence of high affinity sites) to low affinity LBP-1 recognition sites. In addition, this observation correlates with DNase I footprinting analysis showing LBP-1 mediated protection of low affinity sites using either wild type or IS4 promoters or promoters containing severe mutations of high affinity LBP-1 recognition sites (data not shown). Overall, our results suggest that LBP-1, most likely through the low affinity LBP-1 binding sites on the HIV-1 promoter, can compromise the processivity of the RNA polymerase and, therefore, restrict the elongation of HIV-1 transcripts in vitro.
In a further analysis we also tested the effect of LBP-1 on the high
levels of Tat-activated transcription observed in nuclear extracts
under conditions (citrate treatment, see above) which lower their
capacity for transcription elongation (but not initiation) on the HIV-1
promoter(18) . Somewhat surprisingly, LBP-1 suppressed Tat
function under these conditions, but did not further decrease basal
activity (data not shown), indicating that combined effects of LBP-1
and citrate treatment may modify the transcription complex in such a
way that Tat could no longer make it processive. In addition, while the
elongation restriction effected by citrate does not preclude synthesis
of TAR RNA for Tat function(18) , LBP-1 under these particular
conditions might do so and thus inhibit Tat function indirectly. These
considerations suggest, first, that Tat function may require a narrow
window of conditions (e.g. cellular factor concentrations)
which substantially restrict elongation while still allowing the
formation and the function of a TatTAR complex and, second, that
these processes can involve individual effects of either LBP-1 or other
(co)factors altered by citrate treatment.
This report shows that the cellular factor LBP-1 has the ability to restrict the processivity of RNA polymerase complexes initiated at the HIV-1 promoter and, further, that the HIV-1-encoded Tat protein can partially overcome the LBP-1-induced elongation block. These results are important because recent studies have shown that elongation from the HIV-1 promoter is limited in the absence of Tat, and that LBP-1 sites in the HIV-1 promoter overlap an element (IST) implicated in the generation of short transcripts, whose accumulation in some cases is correlated with restricted elongation in the absence of Tat.
The present results may also be relevant to the function of the IST element, which overlaps the high affinity LBP-1 sites. While a study of the effects of mutations in these sites reportedly eliminated a role for LBP-1 in IST function(35) , this possibility is left open by our demonstration that LBP-1 can still bind to templates bearing the same mutations. On the other hand, the IST also exhibits another function, namely enhanced promoter activity, that has not yet been demonstrated for the recombinant LBP-1 isoform employed here. Moreover, an understanding of the exact relationship between the downstream LBP-1 and IST elements has been difficult because of functional initiator elements that overlap the LBP-1/IST sites(6) . Likewise, dissection of functions of the upstream LBP-1 sites is complicated because of their multiplicity and interspersion among the TATA and Sp1 sites. This multiplicity of LBP-1 sites may provide a functional redundancy that could have precluded their implication in HIV-1 promoter functions in previous studies.
Figure 9:
Models for promoter-proximal events that
govern downstream elongation and termination properties of RNA
polymerase. A, bacteriophage P
` promoter. In
the absence of the
Q protein, E. coli RNA polymerase
initiates and, after a transient pause at +16/+17, elongates
and terminates at downstream terminators. When present,
Q binds
via an upstream recognition element (black box) and, via Nus
A, interacts with a paused RNA polymerase and effects a modification
that permits elongation through downstream terminators. The +2 to
+6 element (striped box) effects RNA polymerase pausing
and a conformational change essential for
Q/Nus A interaction
(reviewed in (21) and (54) ). B, Drosophila hsp70 promoter. In the absence of HSF, RNA
polymerase II initiates transcription but pauses at +23 under the
influence of GAGA factor bound to an upstream element(s) (black
box) and a region (striped box) surrounding the
initiation site. After heat shock, the binding of HSF to upstream sites
(not shown) effects release of the elongation block (reviewed in (21) and (55) ). C, HIV-1 promoter. In the
absence of a strong activator, the binding of LBP-1 to low (striped
squares) and high (solid squares) affinity LBP-1
recognition sites during initiation or early elongation events may
effect RNA polymerase II conformation and/or interactions with
elongation factors. This LBP-1 action renders the elongation complex
nonprocessive and leads to the accumulation of randomly terminated
RNAs. In this model, LBP-1 function may be formally analogous to the
functions of the downstream site (+2 to +6) in the
P
` promoter and GAGA factor/initiation region in the hsp70
promoter, in effecting pausing or an elongation block, respectively.
Just as
Q/Nus A and HSF alter RNA polymerase elongation properties
on the
P
` and hsp70 promoters, respectively, it is
proposed that Tat may alter the elongation restriction imposed on RNA
polymerase by LBP-1 on the HIV-1 promoter (see text for further
discussion).
Thus, during the very early phase of elongation by RNA polymerase II on the HIV-1 promoter (e.g. from a stalled position at +13 in the present analysis), the binding of LBP-1 to the low affinity sites flanking the TATA box, or possibly to the high affinity sites, might prevent productive interactions of cellular factors important for promoter clearance (58) and/or distal elongation(46, 47, 49, 51, 52, 59) , such that the transcription complex is only weakly processive (Fig. 9C). Lu et al.(1993) (34) have also suggested that the TATA box in HIV-1 contributes, in the absence of an strong activator, to the formation of nonprocessive transcription complexes. This model could explain how LBP-1 might restrict HIV-1 elongation in the absence of Tat and yet facilitate a Tat response by allowing TAR RNA formation with concomitant pausing for effective Tat binding and function from a promoter proximal position (Fig. 9C, see below also). Consistent with this model, an effect of Tat in reversing the LBP-1-mediated elongation block in standard (untreated) nuclear extracts was demonstrated (Fig. 8).
On the basis of past and present data, several models for Tat action may be considered, all based on the assumption that at least some fraction of the elongation complexes generated in the absence of Tat are only weakly processive. First (Model A), as in the earliest models, Tat may alter the processivity of the associated elongation complex in a co-transcriptional manner. Second (Model B), TAR-bound Tat in weakly processive complexes may enhance, in a stoichiometric fashion, the processivity of subsequently formed transcription complexes at the time of preinitiation complex assembly or function (``reach back'' model). Third (Model C), TAR-bound Tat in a weakly processive complex may enhance the processivity of the following de novo elongation complex (as in Model B) which in turn provides a recognition site for Tat for alteration of the succeeding complex, and so on. In the latter model, appropriate Tat function might still require transient pausing of an otherwise highly processive complex in the vicinity of the promoter. Possibly relevant to this point, GAGA protein-dependent RNA polymerase pausing on the hsp70 promoter (55) is observed even after induction of elongation by heat shock factor(60) . Clearly, Models B and C can explain how Tat could effect a switch in the elongation properties of a nascent complex (to a more processive form) during the process of initiation complex formation (assembly) or function (initiation or very early elongation events). All models can accommodate roles for promoter-proximal DNA elements and interacting factors, including LBP-1 in the present case, in restricting elongation and/or initiation and setting the stage for Tat function. Models B and C also allow the possibility of Tat effects on the rate of initiation through direct interactions with PIC components(20, 61) . In fact, there could be a causal relationship between effects of Tat on initiation and increased processivity, as has been indicated (13, 62, 63) or predicted (21, 28, 30) for conventional activators acting at 5` sites. This could explain some observed nonadditive effects of upstream activators and Tat(25) .
The present study has defined a potentially important role for an LBP-1 isoform (LBP-1b) in restricting the processivity of RNA polymerase on the HIV-1 promoter. Further studies must determine the molecular targets of LBP-1, the essential LBP-1 binding sites and their relationship to the IST, the functions of individual isoforms of LBP-1, whether LBP-1 is required not only for a general restriction of processivity but also for the Tat activation process per se, and how and at what step Tat overcomes the LBP-1 restriction to processivity.