(Received for publication, January 23, 1996; and in revised form, February 5, 1996)
From the
The assembly of a preinitiation complex containing RNA polymerase II on promoter DNA is a complex process that involves several general transcription factors. Using 5-[N-(p-azidobenzoyl)-3-aminoallyl] photocross-linking, we previously determined the locations of the two large subunits of transcription factor (TF) IIA (A35 and A21), TATA box-binding protein (TBP), RNA polymerase II-associated protein (RAP) 30, and TFIIB along the Ad2 ML promoter. We have now localized TFIIE34 and RAP74 just upstream of the transcription start site. The two subunits of TFIIF, RAP74 and RAP30, cross-linked to nucleotides that probed adjacent spaces on the same face of the DNA helix beginning just downstream of TBP at -19 and extending to -5. Specific photocross-linking of TFIIE34 required the presence TFIIE56. In addition, TFIIE and RAP74 strongly stimulated cross-linking of RAP30 and the large subunits of RNA polymerase II to position -19. Our topological data support the idea that RAP74 and TFIIE34 may be involved in melting of the promoter DNA upstream of the initiation site.
Initiation of transcription by RNA pol ()II is a very
complex process, which involves the general transcription factors
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (reviewed in (1) and (2) ). For genes containing a TATA box,
promoter recognition is achieved through binding of TBP, the TATA
box-binding subunit of TFIID, to the TATA element (reviewed in (3) ). TFIIA and TFIIB can bind to TBP and assemble onto the
TBP-promoter complex(4, 5, 6, 7) .
Recruitment of RNA polymerase II is mediated by TFIIB and RAP30, the
small subunit of TFIIF, which can directly associate with
TFIIB(7, 8, 9, 10, 11) .
Direct protein-protein interactions between RNA pol II and TBP have
also been reported(12) . RAP74, the large subunit of TFIIF, can
bind to RAP30 and RNA pol II (13) and stabilize the
association of a TBP
TFIIB
RAP30
RNA pol II complex with
promoter DNA(14) . TFIIE and TFIIH can then sequentially bind
to this TBP
TFIIB
TFIIF
pol II
promoter
complex(15, 16) . TFIIE may bind to several general
transcription factors, including TFIIF (17) and
TFIIH(17) , as well as RNA pol II (15) .
For most promoters, a minimal preinitiation complex containing TBP, TFIIB, TFIIF, and RNA pol II can initiate transcription in vitro and synthesize a transcript if the template DNA is supercoiled(18, 19, 20, 21, 22) . Initiation of transcription at the IgH promoter can even occur in the absence of TFIIF(18, 21) . For linear promoters, TFIIE and TFIIH are required for the production of an accurately initiated run-off transcript(20, 22) . These two factors are involved in promoter clearance, probably because they mediate ATP-dependent melting of the template DNA(20, 22) . Recently, Pan and Greenblatt (23) reported that, under certain circumstances, initiation of transcription is limited by melting of the promoter immediately upstream of the transcription start site, and they suggested that efficient melting of the promoter upstream of the position +1 involves an unidentified factor, which may well be TFIIE and/or IIH.
Although several protein-protein interactions
involved in preinitiation complex formation have been described, little
is known concerning the topology of complexes assembled onto promoter
DNA. Using NR photocross-linking, we recently began to
address the physical structure of preinitiation complexes containing
various sets of general transcription factors and RNA pol II. We
localized the relative positions of two subunits of TFIIA (A35 and A21)
as well as those of TBP, TFIIB, RAP30, and the two largest subunits of
RNA pol II on the Ad2ML promoter(14) . TFIIA cross-links to the
coding strand opposite TBP at the TATA box and cross-links upstream of
TBP around position -40. RAP30 cross-links strongly and TBP and
TFIIB weakly to the coding strand just downstream of TBP at position
-19(14) .
We have now localized two additional polypeptides of the general transcription machinery, RAP74 and TFIIE34, along the Ad2ML promoter. Our photocross-linking experiments also provide information on the enzymology of preinitiation complex assembly.
Figure 1:
The photoreactive probes derived from
the Ad2ML promoter. The DNA sequence of the Ad2ML promoter from
-41 to +15 is shown. The TATA element is underlined on the coding strand, and the position of the transcription start
site (+1) is indicated by an arrow. Mutations (N to T)
used in some of our probes are indicated by dots. Mutations in
the TATA box (T to G and A to G) used in some of our photoprobes are
also shown. The oligonucleotides that were used to prime the synthesis
of each of our 5 photoprobes are represented as open boxes that were aligned with their corresponding sequence on the top or
the bottom strand of the promoter. An arrow within the box indicates the direction in which synthesis is to occur. The
different probes are identified using the positions in between which
the photoreactive nucleotide(s) NR-dUMP (U) are
incorporated. In each case the positions of the
P-radiolabeled nucleotide are marked with an asterisk (*).
Figure 2: TFIIE34 specifically cross-links the Ad2 ML promoter in a region between -14 and -2 in the presence of TFIIE56. A, cross-linking reactions with photoprobe -14/-2 containing either a wild type (lanes 1 and 2) or a mutated (Mut) (lane 3) TATA box (see Fig. 1) were performed with TFIIB, RAP74/30, RNA pol II, and TFIIE56/34 in the presence (lanes 1 and 3) or in the absence (lane 2) of TBP. The positions of molecular weight standards are indicated. B, photocross-linking experiments using various combinations of TBP, TFIIB, RAP74/30, RNA pol II, and TFIIE56/34 were performed with probe -14/-2 (lanes 1-4). The products of some reactions performed using all the factors were immunoprecipitated with an antibody directed against TFIIE34 (lane 5) or a control antibody (lane 6). The position of TFIIE34 (E34) is indicated.
Surprisingly, we have not yet been able
to cross-link TFIIE56 to any of our photoprobes in the presence of TBP,
TFIIB, RAP30/74, RNA pol II, and TFIIE56/34. This was particularly
surprising since TFIIE is a heterotetramer containing 2 molecules of
TFIIE56(31) . Our photocross-linking data suggest that the
large subunit of TFIIE may be located quite far away from the promoter
DNA or in a region where the chemical arm of NR-dUMP cannot
reach. Alternatively, TFIIE56 may act as a molecular chaperone in
catalyzing the assembly of the preinitiation complex in the presence of
TBP, TFIIB, TFIIF, RNA pol II, and TFIIE34. If this is true, TFIIE56
may become stably bound to the preinitiation complex only in the
presence of TFIIH.
Figure 3: RAP74 cross-links to position -15 and -5, but not to position -10. Photocross-linking experiments in the presence of various combinations of TBP, TFIIB, RAP74/30, RNA pol II, and TFIIE56/34 (lanes 1-3) were performed using probes that place a photoreactive nucleotide at positions -15 (A), -10 (B), and -5 (C) (see Fig. 1). In each case, one reaction performed in the presence (lane 4) or in the absence (lane 5) of TBP was immunoprecipitated using an antibody raised against RAP74. The positions of RNA pol II larger subunits (Pol II) and RAP74 as well as those of molecular weight standards are indicated.
Figure 5:
Schematic representation of a
TBPTFIIA
TFIIB
TFIIF
RNA pol II
TFIIE
complex. The structure of the TBP-promoter complex is derived from
x-ray crystallographic data on a complex containing the Ad2ML promoter
and Arabidopsis thaliana TBP(35, 36) . The
predicted positions of TFIIA(14) , TFIIB(14) , RAP74,
RAP30, and TFIIE34 are as predicted by our photocross-linking data.
Nucleotides -19 (coding strand) and -15 and -5
(non-coding strand) that cross-link to RAP30 and RAP74 are highlighted
in red. RNA pol II is not shown because our data on the
relative positions of various subunits are still too
fragmentary.
Figure 4: TFIIE and RAP74 stimulate cross-linking of RAP30 and RNA pol II to position -19. A, photocross-linking experiments with probe -19 (see Fig. 1) used various combinations of TBP, TFIIB, RAP74/30, RNA pol II, and TFIIE56/34 (lanes 1-4). The positions of RAP30 and RNA pol II subunits (Pol II) as well as that of molecular weight standards are indicated. B, photocross-linking reactions with probe -19 in the presence (lanes 3, 5, 7, and 9) or in the absence (lanes 4, 6, 8, and 10) of TBP were immunoprecipitated using antibodies raised against RAP30 (lanes 3 and 4), TFIIB (lanes 5 and 6), TBP (lanes 7 and 8), and TFIIE34 (lanes 9 and 10). The positions of RAP30 and TFIIE 34 (E34) are indicated.
Fig. 5shows a schematic representation of our photocross-linking data in the context of a molecular structure for the TBP-promoter complex(35, 36) . We propose that position -19 delimits the space where RAP30 and TFIIB stabilize the interaction between TBP and RNA pol II along the promoter DNA. The position shown for TFIIB is based on our cross-linking experiments performed in the absence of TFIIE(14) . RAP30 and RAP74 are placed along one face of the DNA between -19 and -5, and TFIIE34 is located between -14 and -2.
Recently, Pan and Greenblatt (23) reported that initiation of transcription on closed, linear templates is limited by melting of the promoter immediately upstream of the transcription start site. Synthesis of a promoter-specific trinucleotide on linear DNA requires TFIIF and is stimulated by TFIIE(23) . Artificial melting of the promoter DNA between -9 and -1 on such a template obviates the requirement for TFIIF, suggesting that TFIIF may be involved in melting of the promoter upstream of +1(23) . Photocross-linking of RAP74 and TFIIE34 to nucleotides located between -15 and -2 suggests that these factors may be involved in melting of the DNA helix during transcription initiation.