(Received for publication, August 31, 1995; and in revised form, December 5, 1995)
From the
A double-stranded RNA structure transcribed from the HIV-1 long
terminal repeat known as TAR is critical for increasing gene expression
in response to the transactivator protein Tat. Two cellular factors,
RNA polymerase II and TRP-185, bind specifically to TAR RNA, but
require the presence of cellular proteins known as cofactors which by
themselves are unable to bind to TAR RNA. In an attempt to determine
the mechanism by which these cofactors stimulate binding to TAR RNA, we
purified these factors from HeLa nuclear extract and amino acid
microsequence analysis performed. Three proteins were identified in the
cofactor fraction including two previously described proteins,
elongation factor 1 (EF-1
) and the polypyrimidine
tract-binding protein (PTB), and a novel protein designated the
stimulator of TAR RNA-binding proteins (SRB). SRB has a high degree of
homology with a variety of cellular proteins known as chaperonins.
Recombinant EF-1
, PTB, and SRB produced from vaccinia expression
vectors stimulated the binding of RNA polymerase II and TRP-185 to TAR
RNA in gel retardation analysis. These studies define a group of
cellular factors that function in concert to stimulate the binding of
TRP-185 and RNA polymerase II to HIV-1 TAR RNA.
The regulation of HIV-1 gene expression is dependent on a number of cis-acting regulatory elements in the long terminal repeat(1) . Cellular factors binding to each of these elements are important for the assembly of transcription complexes that are responsive to the transactivator protein Tat. Mutagenesis of the HIV-1 long terminal repeat has demonstrated that the SP1(2, 3) , TATA(4, 5, 6, 7) , and TAR (8, 9, 10, 11, 12) elements are each critical for Tat activation. The TAR element which extends between +1 and +60 appears to be the major regulatory element required for Tat transcriptional activation(4, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) . Thus, to determine the mechanism of Tat activation will require a better understanding of the interplay of these different regulatory elements.
The TAR element when inserted downstream of a variety of heterologous viral and cellular promoters will confer on these promoters the ability to be activated by Tat(4, 9, 13, 18) . This suggests that TAR is necessary and, in many cases, sufficient for Tat activation although the TATA and SP1 sites are also critical in modulating the degree of Tat activation. TAR DNA binds a variety of proteins including UBP/LBP-1 (22, 23, 24) , YY-1(25) , and TDP-43 (26) and induces the synthesis of short or nonprocessive transcripts in both the HIV-1 long terminal repeat and heterologous promoters(18, 27) . Although TAR DNA likely plays a regulatory role in the control of HIV-1 gene expression, it appears that the ability to form a stable RNA stem-loop structure transcribed from TAR and extending between +1 and +60 is critical for Tat activation(10, 12, 15, 20, 21) . Two elements within TAR RNA, a three-nucleotide bulge structure (15, 28, 29, 30, 31) and a six-nucleotide loop sequence(10, 11, 15, 30, 32) , are both critical for Tat activation. This is likely due to the ability of the bulge to serve as the binding site for Tat(28, 29, 30, 31, 33, 34) and the loop to serve as the binding site for a cellular factor designated TRP-185 or TRP-1(32, 35) . Although mutagenesis of the loop or the bulge markedly reduce Tat activation, the mechanism by which the binding of either TRP-185 or Tat to TAR RNA stimulates subsequent transcriptional activation remains to be determined.
Recently, we demonstrated that, in addition to Tat and TRP-185, RNA polymerase II is also able to bind specifically to TAR RNA(36) . HIV-1 templates with wild-type TAR elements that exhibit high in vivo levels of activation in response to Tat bind RNA polymerase II with high affinity, whereas HIV-1 templates with mutated TAR elements that bind RNA polymerase II with lower affinity are not markedly activated in vivo by Tat(36) . During the process of transcriptional elongation in both eucaryotic and procaryotic promoters, RNA polymerase II likely has separate binding sites for both DNA and RNA(37, 38, 39, 40) . Thus, one mechanism to explain the function of TAR in the activation of HIV-1 gene expression would be that the TAR RNA loop and bulge elements bind cellular and viral proteins, respectively, that are each able to help disengage RNA polymerase II bound to TAR RNA. Whether either or both of these factors are able to stably bind to the RNA polymerase II during subsequent transcriptional elongation remains to be determined.
Both RNA polymerase II and TRP-185 by themselves bind weakly to TAR RNA in gel retardation assays(32, 35, 36) . However, the addition of another groups of proteins purified from HeLa nuclear extract markedly stimulates both RNA polymerase II (36) and TRP-185 binding to TAR RNA (32, 35) . These proteins, designated cofactors, are alone unable to stably bind to TAR RNA. The mechanism by which these factors stimulate TRP-185 and RNA polymerase II binding to TAR RNA is unclear. Possibilities include post-translational modification of either the TRP-185 or RNA polymerase II or direct interaction of the cofactors with either TRP-185 or RNA polymerase II bound to TAR RNA . To address these possibilities, we purified proteins in the cofactor fraction and subjected them to amino acid microsequence analysis. We were able to isolate the cDNAs encoding three cellular proteins present in the cofactor fraction. These cDNAs were placed into vaccinia expression systems to produce these recombinant proteins in HeLa cells. Addition of the three purified recombinant cofactor proteins markedly stimulated the binding of both RNA polymerase II and TRP-185 to TAR RNA. Our results suggest that the cofactors function to stimulate TRP-185 and RNA polymerase II binding by both direct interactions with proteins bound to TAR RNA and also by potentially modifying the structure of these proteins. The cofactors potentially have a novel regulatory mechanism which may be involved in the control of HIV-1 gene expression.
Western blot analysis was performed using the 12CA5 monoclonal
antibody (47) and ECL reagents (Amersham). The amounts of
protein used in Western blot was 300 ng of each of the recombinant
proteins purified using nickel chromatography. Recombinant EF-1,
PTB, and CF58 had molecular masses of 54, a doublet of 58, and 62 kDa,
respectively.
In an attempt to determine the mechanism by which these cofactors stimulated the binding of TRP-185 and RNA polymerase II to TAR RNA, we purified the proteins responsible for this cofactor activity. The ability of the cofactors to stimulate the binding of recombinant TRP-185 to TAR RNA in gel retardation studies was used as our assay. To purify the cellular cofactors, HeLa nuclear extract prepared from 60 liters of cells was applied to a heparin agarose column. The cofactor activity was eluted with 0.4 M KCl, applied to a hydroxylapatite column, and then eluted with 0.1 M potassium phosphate. Following ammonium sulfate precipitation and chromatography on a Superdex 200 column, the active fractions were pooled and applied to a Q-Sepharose column. The flow-through fractions were further fractionated on a Bio-Rex 70 column, and the protein fractions which stimulated the binding of recombinant TRP-185 to TAR RNA were pooled. A flow chart of this purification scheme is shown in Fig. 1.
Figure 1: Purification scheme of cellular cofactors from HeLa cells. The protocol for the fractionation of HeLa cell nuclear extract to purify the cellular cofactors which stimulate TRP-185 binding is shown. The numbers in the figure indicate the concentration of KCl used to elute each column with the exception of the hydroxylapatite Bio-Gel column in which the concentration of potassium phosphate is indicated.
The purified cofactor fraction was then assayed for its ability to stimulate TRP-185 binding to TAR RNA. Addition of increasing amounts of recombinant TRP-185 alone resulted in only minimal binding to TAR RNA (Fig. 2, lanes 2-4). However, upon the addition of the purified cofactors to TRP-185, there was a marked increase in its binding to TAR RNA (Fig. 2, lanes 5-7). There was no binding of the cofactor fraction alone to TAR RNA (Fig. 2, lane 1). The enhancement of TRP-185 binding by the cofactors was not seen with equivalent amounts of other proteins such as GST or albumin (Fig. 2, lanes 8-11). Finally, it was found that increasing the amount of cofactors from 0.1 to 1.0 µg markedly increased the binding of TRP-185 to TAR RNA (Fig. 2, lanes 12-14). These results indicate that the purified cellular cofactors did not bind directly to TAR RNA by themselves, but acted to markedly stimulate the binding properties of TRP-185 to TAR RNA.
Figure 2: Cellular cofactors are required for TRP-185 binding activity to HIV-1 TAR RNA. Gel retardation analysis was performed with a labeled HIV-1 TAR RNA probe (lane 1) in the presence of increasing amounts of purified recombinant TRP-185, 10 ng (lane 2), 25 ng (lane 3), 50 ng (lane 4) either alone or in the presence of 0.4 µg of purified cellular cofactors (lanes 5-7). The enhancement of TRP-185 binding was assayed with wild-type TAR RNA and TRP-185 alone (50 ng) (lane 8) or with 0.4 µg of either cellular cofactors (lane 9), bovine serum albumin (lane 10), or GST (lane 11). TRP-185 binding to TAR RNA was assayed by adding increasing amounts of purified cellular cofactors 0.12 µg (lane 12), 0.4 µg (lane 13), or 1.0 µg (lane 14) and comparing this to the addition of 1 µg of bovine serum albumin (lane 15).
The third protein of 58 kDa generated two peptides with the amino acid sequences VVSQYSSLLSPMS and AFADAMEVIPSTLAENAGLNPISTV. Sequence comparison of these two peptides demonstrated no sequence homology with other proteins in GenBank. Degenerate oligonucleotides were synthesized to the 5` and 3` portions of the 25-amino acid peptide obtained from this 58-kDa protein, and these primers were used in polymerase chain reaction analysis with HeLa cDNA. A 75-base pair fragment was obtained and DNA sequence analysis confirmed that the deduced amino acid sequence matched the amino acid sequence obtained by microsequence analysis. This fragment was then used to screen a HeLa cDNA library and resulted in the isolation of a 2.0-kb cDNA which contained a 539-amino acid open reading frame that we designated stimulator of TAR RNA-binding proteins (SRB).
Figure 3: Amino acid sequence of SRB. A, the deduced amino acid sequence of the SRB cDNA is indicated with the position of the two peptides generated from the Lys C digestion of the purified SRB protein underlined. The position of a conserved 80-amino acid region in the amino-terminal portion of SRB which is related to the chaperonin family is demarcated by a box as is another seven-amino acid domain which has homology to the P loop sequence which serves as potential binding site for ATP or GTP. B, the homology of the amino terminus of SRB with the previously characterized ANC2 protein is indicated.
Figure 4:
Northern analysis of the expression of
cellular cofactors. A blot containing 2 µg of poly(A)-selected RNA
from a variety of derived human tissues was analyzed sequentially with
cDNA probes derived from either EF-1 (A), PTB (B), SRB (C), or glyceraldehyde-3-phosphate
dehydrogenase (D) and subjected to autoradiography. The lanes
include a size marker (lane M) and RNA isolated from tissues
including heart (lane 1), brain (lane 2), placenta (lane 3), lung (lane 4), liver (lane 5),
skeletal muscle (lane 6), kidney (lane 7), and
pancreas (lane 8).
Figure 5:
Western blot analysis of recombinant
cofactor protein. Each of the cellular cofactors was expressed in HeLa
cells following transfection of their respective cDNAs cloned into the
PTM1 expression vector and infection with recombinant vaccinia virus
producing T7 polymerase. Each of these histidine-tagged proteins was
purified using nickel affinity chromatography, and Western blot
analysis was performed with 300 ng of each protein and detected using
12CA5 monoclonal antibody. This monoclonal antibody recognizes the
influenza hemagglutinin epitope inserted on the carboxyl terminus of
SRB (lane 1), PTB (lane 2), and EF-1 (lane
3). The position of the molecular mass markers (MW) is
also indicated.
Figure 6:
Recombinant cofactors stimulate TRP-185
binding to TAR RNA. Gel retardation was performed with
vaccinia-produced recombinant TRP-185 in the presence of different
recombinant cofactors. A, the wild-type TAR RNA probe was
incubated with 50 ng of EF-1, PTB, and SRB (lane 1), with
50 ng of TRP-185 either alone (lane 2) or in the presence of
50 ng of either EF-1
(lane 3), PTB (lane 4), or
SRB (lane 5) or both 50 ng of EF-1
and PTB (lane
6), EF-1
and SRB (lane 7), or PTB and SRB (lane
8). B, increasing amounts (10, 20, or 50 ng) of a
combination of EF1-
, PTB, and SRB (lanes 1-3) were
incubated with TRP-185, and the binding was compared to that with 0.4
µg of native cofactors purified from HeLa cells (lane
4).
Finally, we tested whether the addition of all three cofactors was able to increase the binding of TRP-185 to TAR RNA. Addition of increasing amounts of all three cofactors resulted in a marked stimulation of TRP-185 binding to wild-type TAR RNA (Fig. 6B, lanes 1-3). This binding was equivalent to the maximal binding of TRP-185 seen in the presence of native cellular cofactors purified from HeLa cells (Fig. 6B, lanes 3 and 4). These results indicate that the addition of the recombinant cofactors will completely reconstitute TRP-185 binding. Since the native cellular cofactors in HeLa cells exist as a complex during purification, the ability of the individual recombinant cofactors to result in low level stimulation of TRP-185 binding to TAR RNA is likely due to cross-contamination with other copurifying cofactors.
Figure 7:
Recombinant cofactors stimulate RNA
polymerase II binding to TAR RNA. A, gel retardation was
performed with wild-type TAR RNA (lane 1) and 50 ng of
purified RNA polymerase II alone (lane 2) or in the presence
of 50 ng of either EIF-1 (lane 3), PTB (lane 4),
or SRB (lane 5) or both EF-1
and PTB (lane 6),
EF-1
and SRB (lane 7), or PTB and SRB (lane 8). B, increasing amounts (10, 20, or 50 ng) of a combination of
each of the three cofactors EIF-1
, PTB, and SRB (lanes
1-3) were incubated with RNA polymerase II, and the binding
to TAR RNA was compared with that of 0.4 µg of native cofactors (lane 4). The binding properties of a combination of 50 ng of
EF-1
, PTB, and SRB (lane 5) or 0.4 µg of purified
cellular cofactors in the absence of added RNA polymerase II is also
shown (lane 6).
To address whether the
cofactors directly associated with proteins bound to TAR RNA during gel
retardation analysis, we raised rabbit polyclonal antibodies to
glutathione S-transferase protein fusions comprised of
portions of either EF-1, PTB, or SRB. Western blot analysis was
performed with each of these polyclonal antibodies, and they each
reacted specifically with vaccinia-produced recombinant cofactors (data
not shown). Each of these antibodies was then affinity-purified using
protein A-Sepharose columns and tested for its ability to supershift a
complex comprised of TRP-185 or RNA polymerase II and cofactors in gel
retardation analysis. Such a result would indicate that the individual
cofactors were present in the TAR RNA complex with either TRP-185 or
RNA polymerase II.
Gel retardation analysis was performed with
recombinant TRP-185 and either the native cofactors (Fig. 8A, lanes 1-6) or the recombinant
cofactors (Fig. 8A, lanes 7-11). Gel
retardation analysis indicated that a complex formed by the addition of
TRP-185 and purified cellular cofactors was supershifted by a
monoclonal antibody directed against TRP-185 (Fig. 8A,
lane 3). Antibody directed against either EF-1 (Fig. 8A, lane 4) or SRB (Fig. 8A, lane 6) did not result in a similar
supershifted complex. However, the addition of antibody directed
against PTB resulted in a shift of the complex bound to TAR RNA (Fig. 8A, lane 5) similar to that seen with
TRP-185 monoclonal antibody. Addition of the PTB antibody alone with
wild-type TAR RNA did not result in a detectable complex in gel
retardation analysis (data not shown).
Figure 8:
PTB associates with TRP-185 but not RNA
polymerase II in a complex with HIV-1 TAR RNA. A, gel
retardation assays were performed with wild-type TAR RNA (lane
1) and recombinant TRP-185 reconstituted with either the native
cofactors purified from HeLa cells (lanes 2-6) or the
recombinant cofactors (lanes 7-11). TRP-185 with either
cellular cofactors (lane 2) or recombinant cofactors (lane
7) in the absence of added antibody or in the presence of 1 µg
of affinity-purified rabbit polyclonal antibody directed against either
TRP-185 (lanes 3 and 8), EF-1 (lanes 4 and 9), PTB (lanes 5 and 10), or SRB (lanes 6 and 11) is shown. B, gel
retardation assays were performed with wild-type TAR RNA (lane
1) and purified RNA polymerase II (50 ng) in the presence of 0.4
µg of native cofactors alone (lane 2) or in the presence
of 1 µg of affinity-purified antibodies directed against the
carboxyl-terminal domain of RNA polymerase II (lane 3),
EF-1
(lane 4), PTB (lane 5), or SRB (lane
6).
These gel retardation
experiments were then repeated using recombinant cofactors and TRP-185.
Antibodies directed against either TRP-185 (Fig. 8A, lane 8) or PTB (Fig. 8A, lane 10)
were again able to supershift the complex bound to TAR RNA, while
antibodies directed against either EF-1 or SRB (Fig. 8A, lanes 9 and 11) were unable
to supershift this complex. These results indicated that PTB was able
to associate directly in a complex comprised of TAR RNA and TRP-185. We
cannot definitively rule out that either EF-1
or SRB may also
associate directly with this complex. This is because only a subset of
antibodies directed against the same protein are able to alter the
mobility of the gel-retarded complex resulting in either a loss or a
shift in the complex. This was true with a panel of rabbit polyclonal
antibodies directed against TRP-185 of which only two of five
antibodies was able to supershift this protein in gel retardation
analysis with TAR RNA (data not shown).
Figure 9:
ATP
inhibits TRP-185 binding to TAR RNA. Gel retardation analysis was
performed with TRP-185 and a mixture of 50 ng of the three recombinant
cofactors EF-1, PTB, and SRB and a wild-type HIV-I TAR RNA probe.
The gel retardation assays included TRP-185 alone (lane 1) or
TRP-185 in the presence of either untreated recombinant cofactors (lane 2), these same cofactors treated with hexokinase (4.5
units) and glucose (250 µM) (lane 3), or ATP (2
mM) (lane 4) prior to incubation with TRP-185.
Cofactors alone in the absence of added TRP-185 which were treated with
either hexokinase and glucose (lane 5) or ATP (2 mM) (lane 6) were also tested in the gel retardation
assay.
The TAR element is critical for the activation of HIV-1 gene expression by the transactivator protein Tat although the mechanism by which TAR functions in conjunction with Tat remains open to question (9-13, 15, 18, 30, 32, 60-62). However, it is clear that the structure of nascent TAR RNA is critical for Tat function(60) . A number of previous studies have demonstrated that the TAR RNA bulge element (15, 28, 29, 30, 31) is important for Tat activation and that the Tat protein is capable of binding to the HIV-1 TAR RNA bulge element (28, 29, 30, 31, 33, 34) . However, mutation of the TAR RNA loop sequences also markedly decrease Tat activation(10, 11, 15, 30, 32) , and, yet, these mutations have little or no effect on Tat binding, suggesting a critical role for cellular factors that bind to TAR RNA loop sequences (28, 29, 30, 31, 33, 34, 43) . We were interested in defining cellular proteins that bind to TAR RNAs transcribed from HIV-1 templates that were activated in vivo by Tat but did not bind efficiently to HIV-1 templates that were not activated in vivo by Tat. Using a biochemical fractionation of HeLa nuclear extract, we determined that only two proteins, TRP-185 and RNA polymerase II, met this criteria(36) .
While characterizing the binding of TRP-185 and RNA polymerase II to TAR RNA, we found that their binding was stimulated greatly by the addition of a group of cellular factors designated cofactors(32, 36) . Although the cofactors by themselves did not exhibit any binding to TAR RNA, the addition of these factors with TRP-185 or RNA polymerase II markedly stimulated their binding to TAR RNA. Previous data suggested that TRP-185 and the cofactors remained stably associated following column chromatography(32) . The ability of the three cofactor proteins to remain associated in the purification scheme used in the current study further suggest that these proteins may exist in a complex within the cell. Whether any cellular RNAs may be associated with this complex remains to be determined. It will be critical to determine whether the cofactors modulate HIV-1 gene expression. However, given the high endogenous levels of these proteins in the cell, transfection assays did not allow us to ascertain the effects of these factors on HIV-1 gene expression.
The purification of the cofactor proteins and
subsequent microsequence analysis revealed that the cofactors were
comprised of at least three proteins. These included EF-1, PTB,
and SRB. The potential relationship of these proteins remains unclear
at this time. EIF-1
is a 53-kDa cytosolic protein which is
important in initiating eucaryotic
translation(43, 44) . EF-1
has been demonstrated
to bind to aminoacyl-tRNA and also bind and hydrolyze GTP. Whether the
hydrolysis of GTP is critical for its ability to stimulate TRP-185 or
RNA polymerase II binding to TAR RNA remains to be determined. It will
also be critical to determine how this primarily cytoplasmic localized
protein is able to be transported and function in the nucleus. It has
been demonstrated previously that several translation factors including
eIF-4F, eIF-4E, and eIF-2 can be detected in the nucleus(63) .
The PTB protein was first purified from nuclear extract and demonstrated to bind to the polypyrimidine tract present in the 3` portion of RNA splice sites(45) . The PTB gene gives rise to alternatively spliced mRNAs which encode proteins with domains homologous to previously described ribonucleoprotein binding domains that mediate binding RNA(45) . It has also been demonstrated that PTB is able to bind directly to the bulge region of the poliovirus RNA leader sequence which is involved in facilitating internal ribosomal initiation(50) . It is interesting to note that this poliovirus RNA sequence contains several three-nucleotide bulge sequences which are identical with the TAR RNA bulge sequences. Using an RNA selection procedure to determine the optimal binding sites for PTB, it has been demonstrated recently that PTB binds preferentially to uridine-rich RNA sequences(51) . Since we have demonstrated that PTB associates with TRP-185 in a complex bound to TAR RNA, it is possible that PTB may in fact be in contact with the TAR RNA bulge. Such a possibility may be one explanation for the failure of TRP-185 to bind to TAR RNA which contains a deleted TAR RNA bulge. However, we have been unable to detect direct binding of PTB to TAR RNA in the absence of either TRP-185 or RNA polymerase II.
The SRB protein
appears to have the greatest effect of the three cofactor proteins on
the binding of TRP-185 and RNA polymerase II to TAR RNA. SRB has
homology to both eucaryotic and procaryotic chaperonin proteins (52, 53, 54, 55) and contains a
so-called P-loop domain (56, 57, 58) which in
a variety of proteins including chaperonins has been demonstrated to
bind ATP or GTP. However, we detected decreased binding of TRP-185 in
the presence of cofactors when either ATP or GTP was added to the gel
retardation assay. It is interesting to note that in contrast to a
variety of other chaperonin proteins which are exclusively cytoplasmic (52, 53) SRB is present in both the nucleus and
cytoplasm. Given the demonstration that the procaryotic chaperonin
protein ClpX is able to stimulate the binding properties of 0 to
its DNA recognition site (59) , it seems likely that other
nuclear chaperonin proteins will be identified that are able to
stimulate the binding of either DNA or RNA-binding proteins to their
cognate binding sites. Since chaperonin proteins exist in heteromeric
complexes with other proteins(53) , it is possible that SRB
forms a hetero-oligomeric complex associated with PTB and EF-1
.
The question arises how the three cofactors are able to stimulate
the binding of TRP-185 and RNA polymerase II to TAR RNA. Both PTB (45, 50, 51) and EF-1 (64) have
been demonstrated to bind directly to other RNAs. The demonstration
that PTB was present in a complex with TRP-185 bound to TAR RNA
suggests that the cofactors may be able to interact either directly
with TRP-185 or bind weakly to a portion of TAR RNA in conjunction with
TRP-185. We have been unable to demonstrate direct interactions between
TRP-185 and any of the cofactors using the mammalian two-hybrid system
so that we would favor the possibility that PTB binds weakly to TAR RNA
in conjunction with TRP-185. The inability to detect PTB in a complex
bound to TAR RNA with RNA polymerase II may be due to the fact that
domains of PTB are no longer accessible to the antibody. It is also
possible that PTB may have a different mechanism of action to stimulate
TRP-185 binding to TAR RNA as compared to that of RNA polymerase II.
The role of EF-1
on stimulating the binding of TRP-185 and RNA
polymerase II to TAR RNA remains unclear. EF-1
like PTB may
potentially bind weakly to TAR RNA in the presence of TRP-185 or RNA
polymerase II but fail to be recognized in a complex bound to TAR RNA
by the antibodies that we have generated. Since EF-1
has been
demonstrated to bind to aminoacyl-tRNA, it seems possible that it may
be capable of binding to TAR RNA in conjunction with other proteins.
Whether the ability of EF-1
to hydrolyze GTP may be important in
either TAR RNA binding or transcriptional regulation remains to be
determined. It is possible that the function of SRB to stimulate
protein binding to TAR RNA (45, 50, 51) is
due to its ability to result in optimal folding of TRP-185 and RNA
polymerase II. However, our results using ATP addition and depletion to
the cofactor fraction would also be consistent with a model in which
EF-1
and SRB function to remove ATP which is potentially bound to
TRP-185 to increase its binding to TAR RNA. Mutagenesis of potential
binding ATP sites in TRP-185, SRB, and EF-1
will be required to
address more carefully the role of the cofactors in the stimulation of
TRP-185 and RNA polymerase II binding to TAR RNA.
In summary, we have isolated the cDNAs encoding three cellular factors that copurify from HeLa nuclear extract and facilitate the binding of TRP-185 and RNA polymerase II to TAR RNA. These cofactor proteins are important in regulating the binding of cellular factors to TAR RNA. Whether these factors have a role on the regulation of HIV-1 gene expression in either the presence or absence of Tat remains to be determined. In vitro transcription studies using extracts depleted of individual or multiple cellular cofactors will be required to address the role of these factors on modulating both basal and Tat-induced levels of HIV-1 gene expression.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U38846[GenBank].