(Received for publication, August 9, 1995)
From the
RAP74, the large subunit of human transcription factor IIF (TFIIF), has been analyzed by deletion mutagenesis and in vitro assays to map functional domains. Tight binding to the RAP30 subunit involves amino acids between positions 1-172. Amino acids 1-205 are minimally sufficient to stimulate accurate transcription from the adenovirus major late promoter in an extract system, although C-terminal sequences contribute to activity. A partially masked RNA polymerase II binding domain has been mapped to the C-terminal region of the protein (amino acids 363-444). Sequences near the N terminus and within the central portion of RAP74 affect accessibility of this domain. Extending this domain to 363-486 creates a peptide that binds polymerase and DNA and inhibits transcription initiation in vitro from non-promoter DNA sites. This larger C-terminal domain may modify polymerase interaction with template during initiation and/or elongation of RNA chains.
Human general transcription factor (TF) IIF (RNA
polymerase II-associating protein (RAP) 30/74, FC,
in rat,
Factor 5 in Drosophila, and factor g in yeast) has functions
in both initiation and elongation of RNA chains (1-12; reviewed
in Refs. 13 and 14). The functions of the general factors will be
understood based on their interactions with template, transcript, RNA
polymerase II, other general factors, and regulatory factors.
TFIIF is structurally and functionally related to bacterial sigma factors. In humans TFIIF is a heteromeric factor, consisting of 28-kDa (RAP30) and 58-kDa (RAP74) subunits, that binds directly to RNA polymerase II. The RAP30 subunit binds RNA polymerase II through a domain that is similar in sequence to the polymerase binding domain of bacterial sigma factors(15, 16) . A masked DNA binding domain in the C-terminal region of RAP30 shows additional structural similarity to a DNA binding region of sigma factors involved in contacting the -35 region of bacterial promoters, although the corresponding RAP30 domain is not known to confer promoter recognition specificity to RNA polymerase II(17, 18) .
Whether the RAP74 subunit can also bind directly to RNA polymerase II has not been clearly established, although several observations in the literature indicate such a possibility. RAP74 induces a gel mobility shift in a complex containing promoter DNA, TBP, TFIIB, and RNA polymerase II in the absence of RAP30(19) . RAP74 can also stimulate transcription elongation by RNA polymerase II in a reaction utilizing a 3`-dC-tailed DNA template in the absence of other factors(20) . At least in the presence of template, therefore, RAP74 interacts with transcription complexes, presumably by binding polymerase.
Binding of RAP30 to RNA polymerase II blocks association between polymerase and nonspecific DNA sites, a function associated with bacterial sigma factors(21, 22) . The TFIIF complex additionally dissociates polymerase from DNA sites to which it was previously bound (21, 22) , and this is another similarity between TFIIF and sigma factors. The RAP30 subunit by itself does not have this additional capability(21) . Dissociation of polymerase from nonspecific DNA sites promotes association with promoters, and in most in vitro systems, TFIIF must be present to promote stable association of polymerase with the preinitiation complex(7, 8) . The RAP30 subunit has partial function in polymerase entry(7, 23) , but the RAP74 subunit stabilizes assembly and modifies interaction of RAP30 with template (24) .
Several laboratories have reported minimal in vitro systems in which some of the general factors become dispensable for accurate initiation from a promoter. Parvin and Sharp (25) reported that TBP, TFIIB, and RNA polymerase II were sufficient for accurate transcription from the adenovirus major late promoter, using a supercoiled template. Similarly, Usheva and Shenk (26) demonstrated that initiator-binding protein YY1, TFIIB, and RNA polymerase II were sufficient for initiation from the P5 promoter of adenovirus-associated virus, using a supercoiled template. Tyree et al.(19) detected accurate initiation from several promoters in vitro using TBP, TFIIB, RAP30, RNA polymerase II, and supercoiled templates. Supercoiling obviates the requirement of an ATP-dependent step that involves TFIIE and TFIIH(27, 28, 29) . TFIIH includes an ATP-dependent DNA helicase activity that may function to separate DNA template strands during promoter escape by RNA polymerase II(30) . Accurate initiation from linear DNA templates additionally requires TFIIF, TFIIE, TFIIH, and hydrolysis of ATP(27) .
The physiological meaning of these minimal systems is not obvious, but it is clear that accurate initiation can occur in the absence of some general factors and by slightly different mechanisms, depending on the factors present in the complex and the state of the template DNA. In several systems the RAP30 subunit of TFIIF demonstrates initiation functions independent of RAP74, particularly in assembly of the preinitiation complex(7) . RAP74, on the other hand, has been shown to have some elongation functions in the absence of RAP30, although RAP30 also stimulates elongation in conjunction with RAP74. RAP30 has not been shown to stimulate elongation in the absence of RAP74(20, 31) .
In experiments from our laboratory, using an extract system depleted
of TFIIF by immunoprecipitation with anti-RAP30 antibodies, RAP30 was
required for accurate initiation. RAP74 did not stimulate initiation
but was required for very early elongation of the
transcript(32) . In these experiments, transcripts initiated in
the absence of added RAP74 were stably associated with template DNA.
Some other TFIIF-depleted extracts we have made behave somewhat
differently. For these preparations, a small segment from the
N-terminal region of RAP74 (amino acids 1-205) is required to
prevent release of transcripts. ()It is not clear from these
more recent studies whether RAP74 is completely dispensable for
initiation, as indicated by the initial work(32) . It is clear,
however, that RAP74 sequence between amino acids 172-517 is
dispensable for initiation. It is our view, based on these results and
those of others, that RAP30 can, in some cases, provide all of the
initiation functions of TFIIF. RAP74 is required for promoter escape by
RNA polymerase II and, in some cases, to prevent release of newly
initiated transcription complexes. RAP30 likely participates in both
promoter escape and stabilization of newly initiated complexes since
the most important region of RAP74 for these processes is the
N-terminal region, which binds RAP30 ( (36) and this paper).
These partially distinct functions of TFIIF subunits appear to be
influenced by interactions with other general factors and regulators.
Since more defined systems may lack both positive and negative
regulators, extract systems will continue to be of importance in
identifying functions for the general transcription factors.
TFIIF
interacts with TFIIB through the RAP30 subunit, and together these
factors cooperate to bring polymerase into the preinitiation
complex(7, 8, 33) . As discussed above, RNA
polymerase II, TFIIB, and either TBP or YY1 can be sufficient for
accurate initiation from promoters in vitro, indicating that
TFIIB and polymerase might minimally suffice to select transcriptional
start sites(25, 26) . Some sua7 mutants in
the yeast gene encoding TFIIB are altered for selection of
transcriptional start sites(34) . Interestingly, the ssu71 mutants, in the gene encoding a yeast RAP74 homolog, revert normal
start site selection in the sua7ssu71 double
mutant(35) . Thus, the small subunit of TFIIF interacts
physically, and the large subunit interacts genetically, with TFIIB.
Our laboratory has recently demonstrated that the C-terminal region of
RAP74 also binds to TFIIB directly, ()so this genetic
interaction may correlate with physical contact between these general
factors. Based on these observations, there may be significant coupling
of TFIIF and TFIIB function in transcription. TFIIF has functions in
both initiation and elongation of RNA chains. TFIIB has known functions
in initiation but may have unrecognized functions in elongation as
well.
In this report, we have used deletion mutagenesis to map functional domains of human RAP74. We confirm the previous mapping of the RAP30 binding domain(36) . The regions of RAP74 that are required for accurate initiation in a depleted extract system are strikingly different from those required in a more defined system(36) , and indicate interaction of RAP74 with both positive and negative factors in the extract. A partially masked RNA polymerase II binding domain has been identified in RAP74 by deletion of N-terminal and central amino acid sequences. This domain appears to modify template interactions by polymerase.
In addition to the six-histidine extension at the C
terminus of each RAP74 mutant, there are various other amino acid
extensions at one or both termini due to sites of insertion into the
vector. RAP74(1-409) and(363-409) have only an HHHHHH tag
at their C termini. RAP74(1-356) and RAP74(1-296) have
VEHHHHHH at their C termini. RAP74(1-205), RAP74(1-172),
RAP74(1-136), and the internal deletion mutants
RAP74(136-258) and RAP74(
137-356) contain
LEHHHHHH at the C terminus, the same as that of full-length
RAP74(1-517). RAP74(1-75) has AAALEHHHHHH at the C
terminus. All of the N-terminal deletion mutants have the same
C-terminal extension as RAP74(1-517), LEHHHHHH, whereas their
N-terminal extensions vary slightly. RAP74(407-517) has
MASMTGGQQMGRIRINSSSVDKLAAA at the N terminus; RAP74(358-517) has
MASMTGGQQMGRIRIRAPSTSS at the N terminus; RAP74(207-517) has
MASMTGGQQMGRIRIRAPSTSC at the N terminus; RAP74(87-517) has only
an additional M at the N terminus. The internal fragments
RAP74(136-258) and RAP74(258-356) both have VEHHHHHH at
their C termini and MASMTGGQQMGRIRIRAPSTSLRP and MASMTGGQQMGRIRIRAPSTSS
at their N termini, respectively. RAP74(363-510) and
RAP74(363-444) have LAAALEHHHHHH at their C termini.
RAP74(363-486) and RAP74(363-452) have KLAAALEHHHHHH at
their C termini.
Proteins were purified by
Ni-affinity chromatography in the presence of 4 M urea, as has been reported for full-length
RAP74(37, 38) . The concentration of mutant proteins
was determined by absorbance at 280 nm using calculated extinction
coefficients based on the content of aromatic amino acids in each
protein. All of the mutants react with polyclonal anti-RAP74 antiserum
except RAP74(258-356) and RAP74(363-409), presumably due to
the absence of epitope (data not shown).
Figure 2:
The
N terminus of RAP74 is involved in RAP30 binding. Histidine-tagged
RAP74 (RAP74-H6) (lane 2) and RAP74 mutants (lanes
3-18) were reconstituted with recombinant RAP30 in
vitro, by dialysis from buffer containing 4 M urea(38) . The resulting complexes were selected on
Ni affinity columns, which bind the histidine tag.
Protein was eluted from the Ni
column with buffer
containing 1% SDS. A, SDS-PAGE gel stained with Coomassie
Brilliant Blue dye. B, Western blot developed with anti-RAP30
antibodies. Recombinant RAP74 without a histidine tag was used in the
reaction shown in lane 1.
RAP74-polymerase binding assays were also done using calf thymus RNA
polymerase II covalently immobilized on an Affi-Gel 10 (Bio-Rad) matrix (1) . Resin with no bound protein ligand served as a negative
control. For each binding assay, 50 µl of resin (100 µg of
immobilized polymerase) was blocked with 0.5 ml of SB containing 0.2%
BSA by rocking at 4 °C for 1 h. About 500 pmol of RAP74 or RAP74
mutant was mixed with the resin and rocked for an additional hour at 4
°C. The mixtures were then washed five times with 1 ml of SB. The
resin was eluted with 40 µl of SB containing 0.5 M KCl and
loaded onto a 16% SDS-PAGE gel. Proteins were visualized by staining
with silver nitrate.
To test whether RAP74 affects initiation or elongation of RNA chains, polymerase was incubated with template at 37 °C for 15 min, NTPs were added, and elongation was continued for 1 h. RAP74 was added to the reaction at the indicated times, either before or after addition of NTPs.
Gel mobility shift assays were
performed according to Killeen and Greenblatt (21) and Conaway
and Conaway (22) with some modifications. The reaction mixtures
(10 µl) contained 20 mM Tris-HCl (pH 8.0), 24 mM HEPES (pH 7.9), 2.4 mM DTT, 40 mM KCl, 11%
glycerol, 0.5 mg/ml BSA, 25 fmol of labeled DNA probe, 0.3 pmol of calf
thymus pol IIA, and RAP74 (0, 0.17, 0.34, 0.51, 0.68, 0.85, and 1 pmol
of RAP74-H6). Reaction mixtures were incubated at 28 °C for 20 min
and immediately loaded onto a 4% polyacrylamide gel containing 0.09%
bisacrylamide, 2.5% glycerol and 0.5 TBE. Electrophoresis was
at 30 mA for 2 h. Dried gels were analyzed by autoradiography.
A set of RAP74 deletion mutants was constructed and analyzed
in binding and functional assays (Fig. 1). Wild type human RAP74
is a 58-kDa protein composed of 517 amino acids. For the most part,
mutants are named according to the amino acids remaining in the
structure. For instance, RAP74(87-517) extends from amino acid 87
to 517 of the wild type protein. Some mutants have been constructed
with internal deletions, and these have been named according to the
amino acids removed from the sequence. For instance,
RAP74(137-356) includes amino acids 1-136 fused to
amino acids 357-517 of the wild type protein. Mutant proteins
were constructed with a C-terminal histidine tag to facilitate
purification and binding assays. In addition, some mutant proteins have
N-terminal extensions. The precise amino acid sequences of mutants are
indicated under ``Experimental Procedures.''
Figure 1: RAP74 deletion mutants. Amino acid sequences included in mutants are indicated by white bars. Mutants were assayed for the following functions: 1) stimulation of accurate transcription (tx.) from the adenovirus major late promoter; 2) binding (bind.) to RAP30; 3) binding to RNA polymerase II (pol II); and 4) inhibition of general transcription by RNA polymerase II. (+) indicates high activity; (±) indicates weakly detected activity; (-) indicates no observable activity; (n.d.) indicates that no determination was made for a particular mutant.
By this analysis,
amino acids between 1-172 of RAP74 are important for RAP30
binding (lanes 2-7). Further deletion to amino acid 136
abolishes strong RAP30 binding (lane 8). Mutants 74-517 (lane 9) and 87-517 (lane 10) have very weak
interaction with RAP30, indicating that sequences between 1 and 74 and
between 87 and 172 contribute to binding. 136-356 also
showed very weak interaction with RAP30 (lane 16), confirming
that at least part of the binding domain is included between amino
acids 137 and 172. The histidine affinity tag on RAP74 mutants was
necessary for retention of RAP30 on the beads, because RAP74 without
the tag did not retain RAP30 (lane 1).
Figure 3: Accurate transcriptional activity of RAP74 mutants. An extract transcription system was depleted of TFIIF by immunoprecipitation with anti-RAP30 and anti-RAP74 antibodies(37, 38) . Transcriptional activity was reconstituted by addition of recombinant RAP30 (5 pmol) and RAP74 or RAP74 mutants. The template contained the Adenovirus major late promoter digested at position +217 relative to transcription initiation. The 217-nucleotide runoff transcript was quantitated using a PhosphorImager, and accurate transcription reported as percentage of the highest value determined in the experiment.
In contrast, the extract system is very sensitive to
N-terminal deletions. The 74-517 mutant is completely inactive in
reconstituting the depleted extract system. Internal deletion mutants
136-258 and
137-356 are also inactive for
transcription, presumably because of deletion of required sequences
between amino acids 172 and 205 and deletion of a portion of the RAP30
binding domain between amino acids 136-172.
Figure 4:
RAP74 has a masked RNA polymerase II
binding domain near its C terminus. A, SDS-PAGE gel stained
with Coomassie Brilliant Blue dye. Calf thymus RNA polymerase II (27
µg) was mixed with 12 µg of histidine-tagged RAP74 or RAP74
mutant, and complexes were selected on Ni affinity
beads. Proteins retained on beads were eluted with buffer containing 1%
SDS. RNA polymerase II subunits retained on the column are identified
along the left side of the figure. B, SDS-PAGE gel
stained with silver nitrate. RAP74 and RAP74 mutants were tested for
binding to a resin on which calf thymus RNA polymerase II was
covalently immobilized (upper panels) or a negative control
column that contained no protein ligand (bottom panels). RAP74
mutants (363-517, 363-510, and 87-517) show some
nonspecific binding to the negative control beads. HM, high
molecular weight protein size standards. LM, low molecular
weight size standards.
Full-length RAP74
binds to RNA polymerase II, but not as tightly as some mutants from
which N-terminal sequences and sequences from the central portion of
the molecule have been removed. In the data shown in Fig. 4A, polymerase binding is most easily scored by
detection of the 180-, 145-, 36-, 25-, and 18.5-kDa polymerase subunits (39) . A polypeptide of 44 kDa that contaminates this
polymerase preparation may be actin(39) . This and other
contaminating proteins are removed by affinity selection of the
polymerase on immobilized RAP74. Interestingly, two polypeptides (28
and 30 kDa), not known to be polymerase subunits, co-purify by
Ni affinity chromatography with RAP74 mutants,
indicating that these peptides are tightly associated with polymerase.
The mutants that bind polymerase most tightly are 358-517, 363-517, 363-510, 363-486, 363-452, and 363-444. Further deletion to 363-409 abolishes binding. Sequences between amino acids 363-444, therefore, appear to be the most important for polymerase binding. A 407-517 mutant has slightly reduced binding, and a 1-409 mutant has weak binding, indicating that portions of the interaction site can be found both N-terminal to amino acid 409 and C-terminal to amino acid 407.
Accessibility of this C-terminal domain appears to be masked by sequences within the central region but made more accessible by N-terminal sequences. The most significant masking of polymerase binding is seen with RAP74(87-517) and(207-517). Full-length RAP74(1-517), on the other hand, binds relatively tightly to polymerase. Since RAP74(358-517) binds tightly to polymerase, sequences between 207 and 358 appear to be responsible for masking polymerase binding by the C-terminal domain. Sequences from 1 to 87 appear to render the polymerase binding domain more accessible.
Figure 5:
RAP74 inhibits general transcription by
RNA polymerase II. A, calf thymus RNA polymerase II was mixed
with supercoiled plasmid DNA in the presence or absence of RAP74 or
RAP74 deletion mutants. ATP, CTP, GTP, and [H]UTP
were added and transcription continued for 1 h. Incorporation of
[
H]UMP into RNA was quantitated on DE-81 filters
as described under ``Experimental Procedures.'' Transcription
initiation is expected to occur from many sites on the DNA template in
this experiment.
-Amanitin was added at 1 µg/ml in a reaction
otherwise identical to that shown in column 2. Reactions
contained 5 pmol of RNA polymerase II and 50 pmol of RAP74 or RAP74
mutant; (*) indicates that 25 pmol of RAP74 mutant was used in these
reactions. B, RAP74 inhibits initiation of RNA chains. Calf
thymus RNA polymerase II was added to supercoiled plasmid DNA and
incubated for 15 min (addition at t = -15 min).
ATP, CTP, GTP, and [
H]UTP were then added (t = 0 min), and RNA synthesis was continued for 1 h. RAP74
was added to reactions at t = -15, 0, 0.5, or 1
min. Once initiation occurs, complexes become resistant to inhibition
by RAP74. Values are reported as the average of duplicate
determinations. The variation between duplicates observed in
experiments in A and B was typically less than 10%,
always less than 15%, and qualitatively similar with replicate
experiments.
Full-length RAP74 and some RAP74 fragments were found to inhibit
polymerase activity 90-95% (Fig. 5A). Some RAP74
mutants confer moderate inhibition (20-70% inhibition), and
others do not inhibit polymerase at all. Of the RAP74 C-terminal
fragments that bind polymerase tightly, 358-517, 363-517,
363-510, and 363-486 inhibit general transcription strongly
(>75%). Mutants 363-452 and 363-444, that bind strongly,
did not substantially reduce polymerase activity, indicating that
sequences from 452 to 486 are important for inhibition. Mutants
87-517 and 207-517, which are reduced for polymerase
binding but include the tight binding site, moderately inhibit
polymerase. Mutants 136-258 and
137-356 inhibited
polymerase activity substantially (>75%). These mutants include the
polymerase binding region but are missing internal sequences that may
mask the polymerase binding site.
Moderate inhibition was also seen
with mutants 1-409, 1-356, 1-296, 1-205, and
136-258, none of which includes the proposed polymerase binding
domain from 363 to 444. In binding assays, however, each of these
mutants was observed to interact weakly with RNA polymerase II ( Fig. 4and data not shown), indicating that a second domain may
be located in the region between amino acids 136-258 that may
interact with polymerase or DNA to inhibit nonspecific transcription.
Mutants containing sequence between 87 and 258 can bind DNA, as
indicated by a gel mobility shift assay.
RAP74 appears to inhibit initiation of new chains in the nonspecific transcription assay. This is indicated in the experiment in Fig. 5B, in which polymerase was incubated with template DNA for 15 min before addition of NTPs, and transcription was allowed to continue for 1 h. RAP74 was added at the beginning of the preincubation (-15 min), coincident with NTPs (0 min), 30 s after NTPs, or 1 min after NTPs, as indicated in the figure. Since RAP74 gives the greatest inhibition when added before NTPs, inhibition is most likely exerted at the level of chain initiation. When RAP74 is added 1 min after NTPs, no inhibition was observed, indicating that elongation was not inhibited. Since RNA is stable in the presence of RAP74 (addition at t = +1 min), inhibition is not due to a contaminating RNase in the RAP74 preparation.
Figure 6: Interactions between RAP74, RNA polymerase II and DNA as indicated by a gel mobility shift assay. The DNA probe includes sequences upstream from the Adenovirus major late promoter from coordinates -262 to -191 (72 base pairs), 5`-end-labeled at position -262. A, RNA polymerase II (pol II; 0.3 pmol), RAP74(1-517) (0.17-1 pmol; numbers indicate the molar ratio of RAP74/pol II), and probe DNA were combined and electrophoresed, as indicated. Monoclonal antibody against the C-terminal domain (Anti-CTD; 0.7 pmol binding sites) of RNA polymerase II was added to some reactions. Gel mobility shift assays with RAP74(358-517) gave qualitatively similar results (data not shown). The dramatic threshold effect for RAP74 binding to DNA (compare lanes 7 and 9) has been reproducible in multiple experiments, but the explanation for this observation is not known.
The analysis of this experiment is
somewhat complicated because both RAP74 and RNA polymerase II can bind
to DNA independently, and both proteins shift the mobility of the DNA
to similar positions. The native molecular weight of RAP74 is estimated
at 470 kDa by gel filtration (38) , and this may explain why
the mobility shifts induced by RNA polymerase II (500 kDa) and by
RAP74 are so similar.
Comparing lanes 7 and 8 to lane 2, essentially all of the polymerase-DNA complex is supershifted by addition of RAP74, under conditions in which RAP74 does not itself efficiently bind to DNA. The implication of this observation is that RAP74 is bound to RNA polymerase II, rather than both proteins binding independently to the same DNA molecule. This interpretation, of course, is consistent with observations made in the binding experiments shown in Fig. 4, in which RAP74 was shown to bind tightly to polymerase in the absence of DNA. RAP74-induced supershifts of the polymerase-DNA complex are clearly seen in lanes 8 and 10. In lanes 12 and 14, a supershifted species with even slower mobility may represent a DNA probe that has bound to it both a RAP74 and a RAP74-polymerase complex. Clearly, RAP74 does not block association of polymerase with DNA, nor does it dissociate complexes once they have formed, since polymerase-DNA complexes are stable when RAP74 is added to the reaction.
RNA polymerase II used for this experiment was in the IIa form, so it has an intact C-terminal domain (CTD) (polymerase was the kind gift of R. Burgess). To demonstrate that polymerase was a component of shifted complexes, monoclonal antibodies directed against the CTD were added to the reactions shown in lanes 15 and 16. All of the polymerase-dependent mobility shifts were disrupted by addition of antibody, while those dependent on RAP74 alone were not affected (lanes 15 and 17). Disruption of DNA binding by anti-CTD antibody indicates that the CTD is proximal to bound template DNA. This observation is consistent with models for CTD functions in initiation and elongation of transcription(40) . Results of mobility shift experiments were qualitatively very similar with polymerase primarily in the IIb form, that is with the CTD removed by partial proteolysis. As expected, however, anti-CTD antibodies did not affect polymerase IIb-induced mobility shifts (data not shown).
The first report of RAP74 mutagenesis was by Yonaha et al.(36) . In those studies a two-hybrid gene reporter system was used to map regions of RAP74 and RAP30 that interact. They determined that subunit-subunit contact required RAP74 amino acids 62-171. Based on a direct binding assay, we show here that RAP74 amino acids between 1-172 are required for tight binding to RAP30 (Fig. 2). Deletion of amino acids from the N terminus of RAP74(1-73) severely impairs binding. The 2-hybrid system may be somewhat more sensitive than the affinity bead procedure to demonstrate weak interactions.
Yonaha et al. also tested mutant proteins for the ability to support accurate transcription in vitro, using a system of biochemically fractionated components and recombinant TBP. They concluded that amino acids from 73 to 205 and between 435 and 517 are required for full transcriptional activity, and amino acids between 356-435 were essential. In contrast, two reports from other laboratories have demonstrated different requirements for RAP74 sequences in accurate transcription. Joliot et al.(41) showed that amino acids between 1 and 206 could support basal accurate transcription in vitro from the human c-fos promoter, but not transcription activated by the serum response factor. They further showed that 1-356 and 1-296 mutants had decreased activity relative to full-length RAP74, indicating the importance of C-terminal sequences. RAP74 sequence between amino acids 206 and 291 was required to fully support activated transcription by the serum response factor. Using a chimeric TFIIF composed of human RAP30 and Drosophila Factor 5a, Kephart et al.(20) showed that the Drosophila factor could support accurate transcription in the human system. C-terminal sequences contributed to accurate transcriptional activity, but the 575-amino acid protein could be deleted to 1-291 without full loss of activity. In contrast to the results of Yonaha et al.(36) , N-terminal sequences from 1 to 85 were found to be essential for activity. In our study, RAP74 was deleted to 1-205 without full loss of activity, but deletion of N-terminal sequences between 1 and 74 eliminated activity, as observed by Kephart et al.
Accurate transcription involves both initiation and elongation of RNA chains. Several laboratories have presented evidence for RAP74-independent initiation, although most of these reports involve use of supercoiled templates(19, 25, 26, 27) . Our laboratory has presented evidence that initiation can occur from a linear template in an extract system depleted of TFIIF, in the absence of added RAP74(32) . This conclusion, however, has been somewhat controversial, as others using different systems have failed to verify this result. Tan et al.(31) demonstrated that RAP74 was necessary for accurate initiation from a linear template using a transcription system consisting of highly purified and recombinant components. The highly purified system and the TFIIF-depleted extract system appear to differ in this respect.
Additionally, our laboratory has shown that RAP74 prevents
dissociation of newly initiated major late promoter transcripts. Sequences within both the N-terminal and C-terminal regions of
RAP74 contribute to the stability of transcription intermediates.
Sequences between amino acids 409 and 517 are required to prevent
release of about 50% of complexes formed in extracts. Sequences between
136 and 205 are essential to preserve the remaining complexes. We find,
for instance, that the 1-172 mutant, which appears nearly
inactive in runoff transcription (Fig. 3), can support accurate
initiation almost as efficiently as full-length RAP74. Newly initiated
complexes formed in the presence of the 1-172 mutant, however,
are very unstable and dissociate from template. RAP74 may be
dispensable for accurate initiation but, in some cases, necessary for
the stability of the newly initiated complex. In other systems,
additional factors may stabilize complexes in the absence of RAP74, and
in this case, the requirement for RAP74 in promoter escape is observed (32) . It is our view that in extract systems the most
essential functions of RAP74 are for elongation rather than initiation.
In early elongation, RAP74 is required to drive polymerase out of a
pause close to the promoter. RAP74 is also required to prevent
transcript release, although factors present in some extract systems
can replace RAP74 for this function(32) .
Recent cloning of
RAP74 counterparts from Drosophila melanogaster(11) and Saccharomyces cerevisiae(35, 42) allows for comparison of sequences with the expectation
that the most important functional domains may also be the most highly
conserved during evolution. Functional domains of human RAP74 are shown
in Fig. 7A. Multiple sequence alignments are shown that
indicate conservation between RAP74 and related proteins in Fig. 7B. These alignments are based on the method of
hydrophobic cluster analysis (HCA)(43) , which is used to
compare weakly similar sequences using visual pattern recognition to
indicate the most likely alignment. In HCA plots, the sequence is
duplicated and displayed as a two-dimensional representation of an
helix. Hydrophobic amino acids are circled and prolines (stars), glycines (diamonds), serines (boxes with dots), and threonines (boxes) are indicated (Fig. 7C). The most highly conserved sequences in this
evolutionary family are similar to the functional domains mapped in
this work.
Figure 7: Evolutionary conservation of RAP74 functional domains. A, functional domains of human RAP74. Black bars indicate sequences aligned in B. B, multiple sequence alignments comparing human RAP74 (h)(47, 48) , Xenopus RAP74 (x)(49) , Drosophila Factor 5a (dF5a)(11) , and S. cerevisiae Ssu71p/Tfg1p (ySsu71)(35, 42) . Conserved amino acids are indicated in bold type or by underlining. Potential casein kinase II sites are indicated by larger type ``S'' or ``S'' in region II. Stop codons are indicated by *. C, HCA plots (43) are shown for regions I and III of the RAP74 evolutionary family. HCA plots were the basis for the alignments shown in B.
The N-terminal domain between amino acid positions 66 and 187 of human RAP74 is the most highly conserved portion of the molecule. This correlates with our mapping of the RAP30 binding domain between amino acids 1 and 172 (Fig. 2) and the minimal domain for accurate initiation in an extract system between amino acids 1 and 205 (Fig. 3). We also have unpublished data that indicates a DNA interaction site between amino acids 87 and 258 of RAP74. Since D. melanogaster factor 5a can substitute for human RAP74 in the accurate transcription assay in the extract system(20) , many specific amino acid changes are tolerated within this region without full loss of function.
Since accurate transcription assays were done in an extract system, factors that stimulate and inhibit transcription are expected to be present that may not be components of more purified and defined systems. As a result, domains of RAP74 may be dispensable in the extract system that are required for transcription in more defined systems. One explanation for such an observation is that an additional factor in the extract complements or replaces the function of an otherwise necessary RAP74 domain. On the other hand, sequences that are required for function in the extract may be dispensable in more defined systems, for instance, because an inhibitory factor present in the extract requires a RAP74 domain to counteract its effect. If this inhibitor is missing from the more defined system, this domain may become dispensable for transcription.
In a more defined transcription system, amino acids 73-435 were minimally required for transcription(36) . Comparison of their results with ours demonstrates that the N terminus of RAP74 is required for transcription in the extract system but not in a more purified system. As we show here, the N terminus of RAP74 is important for RAP30 binding, so a factor present in extracts but missing in more purified systems may make this subunit contact more important for assembly of transcription complexes. Conversely, the C-terminal domain of RAP74 is required for transcription in the purified system but dispensable in the extract system. A positive transcription factor might replace the function of the C-terminal domain of RAP74 or might obviate its requirement by otherwise stabilizing the transcription complex. Since all regulators of transcription have not yet been identified, characterized, and isolated for in vitro use, comparison of purified and extract systems will continue to be of importance to indicate these functions.
From data not shown in this report, we have identified at least two
sites at which casein kinase II phosphorylates the central domain of
RAP74 in vitro. This central region of RAP74 has
the appearance of a regulatory hinge separating N- and C-terminal
domains, and perhaps phosphorylation is important in regulating or
coordinating the function of these domains. RAP74 is known to be highly
phosphorylated in vivo(1) . Alkaline
phosphatase-treated human TFIIF has decreased affinity for RNA
polymerase II and reduced transcriptional activity in a defined
transcription system(44) . At least two likely casein kinase II
sites are present within this sequence (Fig. 7B; region
II), and the region that includes these sites is conserved in human, Xenopus, and Drosophila proteins, although most of
the surrounding central region is divergent between vertebrates and
flies(11) .
A partially masked RNA polymerase II binding
domain has been located between amino acids 363 and 444 on human RAP74 (Fig. 4). Extending this domain to 363-486 makes it a
potent inhibitor of transcription initiation by RNA polymerase II in vitro from non-promoter DNA sites (Fig. 5). Although
the precise mechanism of non-promoter initiation from a supercoiled
template is not known, these experiments show that binding to this
region of RAP74 influences a specific catalytic function of polymerase.
General transcription inhibition, therefore, is a measure of the
specificity of the interaction between the C-terminal region of RAP74
and RNA polymerase II. The C-terminal domain between 363 and 517 binds
to DNA independent of polymerase in a gel mobility shift assay, so DNA interactions are likely a component of C-terminal domain
function.
Accurate transcription assays in both extract and purified systems indicate the importance of the C-terminal domain in RAP74 function. In our extract system, using the adenovirus major late promoter, RAP74(1-517) has a higher transcriptional activity than RAP74(1-409) (Fig. 3), indicating that this C-terminal domain influences accurate transcription. Using a purified system, Yonaha et al.(36) showed that C-terminal sequences of RAP74 were essential for transcription. In their studies, RAP74(1-435) had decreased activity compared to 1-517. Further deletion to 1-356 abolished activity, demonstrating the importance of the sequence between 356 and 435 in the purified system. Comparing their results to ours, the minimally sufficient sequence for the purified system between 1 and 435 includes most or all of the RNA polymerase II binding domain, but is missing sequences required for inhibition of transcriptional initiation from non-promoter DNA sites (Fig. 4, Fig. 5, and Fig. 7). Further deletion to 1-356 removes the polymerase binding domain and inactivates the protein for transcription in the purified system. When this mutant is tested in the crude extract system, however, it stimulates transcription, as do more radical deletions to 1-205 (Fig. 3). In experiments utilizing the c-fos promoter, RAP74(1-206) was shown to support accurate transcription in vitro in a system using reconstituted fractions and recombinant components(41) , consistent with the results presented here. As mentioned above, deletion to 1-172 supports accurate initiation but not efficient elongation.
The C-terminal region of RAP74 has recently been shown to be important to stimulate a protein phosphatase that dephosphorylates the RNA polymerase II CTD(45) . The function of this phosphatase may be to regulate elongation by reducing CTD phosphorylation and/or to promote polymerase recycling after termination, since polymerase enters the preinitiation complex most efficiently in the dephosphorylated state (40) . Interestingly, the central region of RAP74 masks phosphatase stimulation, and the presence of the N-terminal region decreases masking, as seen in the general transcription inhibition assay described above. Most likely, stimulation of the CTD phosphatase is mediated through the interaction of the C-terminal region of RAP74 with polymerase.
Since binding of RAP74 inhibits transcription from non-promoter DNA sites, this domain appears to modify template contacts by polymerase. A domain of a transcription factor that interacts with RNA polymerase II and DNA might be expected to regulate contacts between polymerase and template at various stages of the transcription cycle. For instance, the C-terminal domain may help release polymerase from nonspecific DNA sites to facilitate termination and polymerase recycling to a promoter. RAP74 is implicated in this function, because TFIIF catalyzes polymerase release from nonspecific DNA sites but RAP30 by itself does not(21, 22) . Such a role for RAP74 is consistent with its function in stimulating the CTD phosphatase, as discussed above. However, RAP74 by itself is incapable of catalyzing polymerase release (Fig. 6). Apparently, both subunits play a role in this process, and although the C-terminal domain of RAP74 may participate in this function, this conclusion cannot be drawn from the available data. RAP74 has been shown to stimulate elongation by RNA polymerase II in the absence of other transcription factors(20) . Potentially, the C-terminal domain of RAP74 could be involved in stimulating polymerization by altering contacts with template. As mentioned above, the C-terminal region contributes to the stability of elongation complexes.
The amino acid sequence between positions 363 and 517 is conserved between human RAP74, Xenopus RAP74, and to a lesser extent Drosophila Factor 5a and yeast Tfg1p/Ssu71p (Fig. 7, B and C). Inspection of the human RAP74 polymerase-binding sequence(363-444) does not reveal a domain with significant hydrophobic structure. This sequence is somewhat serine-, threonine-, proline-, and glycine-rich, but the relationship of these characteristics and polymerase binding is unknown. The sequence from 450 to 517 shows conservation between yeast, Drosophila, and vertebrates, and has significant hydrophobic structure (Fig. 7, B and C). Some of this conserved region (444-486) appears to be required for inhibition of non-promoter initiation by RNA polymerase II (Fig. 5), but sequence from 487 to 517, although conserved, is dispensable for these in vitro assays.
These studies are consistent with the model that RAP74 has N- and C-terminal domains separated by a flexible, highly charged hinge domain. The N-terminal domain binds to RAP30 and has functions in assembly of the preinitiation complex, which are particularly noticeable in highly purified systems. The C-terminal domain interacts with RNA polymerase II and appears to modify polymerase contacts with template and with a CTD phosphatase. The central region may regulate C-terminal domain function, as indicated by masking of RNA polymerase II-binding (Fig. 4), inhibition of non-promoter transcription (Fig. 5), and stimulation of CTD phosphatase(45) . Consistent with regulatory functions for the highly charged central hinge, this is the site for in vitro phosphorylation by casein kinase II. Interactions between the N-terminal and C-terminal regions may occur, because N-terminal sequences reduce masking by central sequences.
Since RAP74 can bind to RNA polymerase II independently from RAP30, RAP74 can enter preinitiation complexes or remain in elongation complexes in the absence of the RAP30 subunit. Independent function of TFIIF subunits, therefore, may be important in regulated initiation or elongation. For instance, RAP30 can stimulate initiation independent of RAP74(19, 32) . On some promoters, therefore, RAP74 could be supplied to the complex after initiation to commence elongation. RAP74 only weakly stimulates elongation independent of RAP30(20, 31) , so this phase of transcription may be regulated by altering subunit contacts. Interactions between subunits may be modified by transcriptional regulators.
The transcriptional activation domain of serum response factor binds to the central region of RAP74(206-291), and this contact is required for enhancement of accurate transcription in vitro from the c-fos promoter(41, 46) . Since this corresponds to the phosphorylated region of RAP74, it will be interesting to determine whether phosphorylation influences regulation by serum response factor. The DNA binding and dimerization domain of serum response factor interacts with the C-terminal region of RAP74 (amino acids 437-517), so this region may receive signals from activators. Since TFIIF is involved both in initiation and elongation, this raises the possibility that control can be exerted during multiple stages of the transcription cycle.