(Received for publication, May 24, 1995; and in revised form, August 9, 1995)
From the
General transcription factor SIII, a heterotrimer of 110-, 18-, and 15-kDa subunits, was shown previously to stimulate the overall rate of RNA chain elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along DNA templates (Bradsher, J. N., Jackson, K. W., Conaway, R. C., and Conaway, J. W.(1993) J. Biol. Chem. 268, 25587-25593). In this report, SIII is shown to possesses the novel ability to direct robust but promiscuous transcription by RNA polymerase II on duplex DNA templates in the absence of initiation factors. Mechanistic studies reveal that SIII promotes RNA synthesis by substantially increasing the efficiency with which RNA polymerase II initiates promoter-independent transcription from the ends of duplex DNA. Remarkably, SIII appears to have a negligible effect on de novo synthesis of end-to-end transcripts. Instead, analysis of reaction products indicates that SIII is capable of promoting a dramatic increase in the ability of RNA polymerase II to extend the 3`-hydroxyl termini of duplex DNA fragments, in a template-directed reaction exhibiting no strong preference for 3`-protruding, 3`-recessed, or blunt DNA ends. Although RNA polymerase II has been shown previously to catalyze primer-dependent transcription, SIII is the first eukaryotic transcription factor found to promote this reaction. Based on these findings, we propose that SIII may suppress transient pausing by RNA polymerase II by helping to maintain the 3`-hydroxyl terminus of the nascent RNA chain in its proper position in the polymerase active site.
A growing body of evidence indicates that the elongation stage of eukaryotic messenger RNA synthesis is a major site for the regulation of gene expression(1, 2) . To date, four transcription factors (SII, SIII, TFIIF, and Tat) that regulate the activity of the RNA polymerase II elongation complex have been defined biochemically. On the basis of their mechanisms of action, these four elongation factors have been assigned to two broad functional classes.
One class, which includes SII and Tat, is composed of transcription
factors that function as ``anti-terminators'' to promote
readthrough by RNA polymerase II through a variety of transcriptional
impediments (3, 4) . SII is a 38-kDa protein that
binds RNA polymerase II and promotes readthrough by polymerase through
specific attenuation sites in such genes as human histone H3.3,
adenovirus 2 major late, and adenosine deaminase, as well as through
some DNA-bound proteins and drugs. SII-dependent readthrough by RNA
polymerase II through these blocks to elongation is accompanied by a
reiterative process of nucleolytic cleavage and re-extension of
portions of the 3`-ends of growing RNA chains. This reiterative process
of shortening and re-extending growing RNA chains appears to be an
obligatory step in readthrough by RNA polymerase II through
SII-sensitive blocks to transcription elongation. Tat is an 10-kDa
protein encoded by the type 1 human immunodeficiency virus (HIV-1). (
)Assisted by one or more cellular transcription factors,
Tat promotes efficient elongation by RNA polymerase II through the HIV
long terminal repeat at least in part through interactions with an RNA
hairpin present in the transactivation response element (TAR) located
in the 5`-untranslated region of the HIV-1 polyprotein gene transcript.
A number of additional attenuation sites that appear to play roles in
regulating gene expression have been identified near the 5`-ends of a
variety of genes including the Drosophila hsp70, hsp26, hsp27,
- and
-tubulin,
polyubiquitin, glyceraldehyde-3-phosphate dehydrogenase, and human
c-myc genes(2, 5) . Neither the transcription
factors nor mechanisms that regulate passage of RNA polymerase II
through these sites have been established.
The second class of
transcription factors known to regulate the activity of the RNA
polymerase II elongation complex includes TFIIF and SIII, whose primary
missions are apparently to boost the overall rate of RNA chain
elongation. Because RNA polymerase II purified from many sources is
unable to catalyze RNA synthesis on naked DNA templates at rates much
greater than 10% of the measured rates of messenger RNA synthesis in vivo(6) , elongation factors like TFIIF and SIII
are likely to play a vital role in gene expression by reducing the
transit time of polymerase over the long stretches of chromosomal DNA
encompassing many eukaryotic protein-coding genes. TFIIF is unique
among general transcription factors by virtue of its ability to
function in both the initiation and elongation stages of transcription (7, 8) . In higher eukaryotes, TFIIF is a heterodimer
of 70-kDa (RAP74) and
30-kDa (RAP30) subunits(8) . Saccharomyces cerevisiae TFIIF is a heterotrimer of
105-kDa, 50-kDa, and 30-kDa subunits; the
105- and 50-kDa
subunits are homologues of RAP74 and RAP30,
respectively(9, 10) . We recently purified elongation
factor SIII to apparent homogeneity from rat liver nuclear extracts (11) . SIII is a heterotrimer of
110-, 18-, and 15-kDa
subunits. Although it is clear that TFIIF and SIII are each capable of
interacting directly with the RNA polymerase II elongation complex and
stongly stimulating the overall rate of RNA synthesis, their mechanisms
of action are poorly understood.
Promoter-specific transcription initiation by RNA polymerase II depends on a set of five general initiation factors referred to as TFIID (which can be replaced by its TATA box binding subunit TBP), TFIIB, TFIIE, TFIIF, and TFIIH(8) . In the absence of these initiation factors, purified RNA polymerase II binds efficiently to single-stranded DNA and initiates transcription, but is not capable of recognizing its promoter or of efficiently initiating transcription on double-stranded DNA templates(12) . In the course of studies investigating the mechanism of SIII action, we discovered that it possesses the ability to direct promoter-independent transcription initiation by RNA polymerase II on duplex DNA templates in the absence of initiation factors. Analysis of the products of this reaction revealed that SIII-dependent transcription results from an SIII-induced increase in the ability of RNA polymerase II to extend the 3`-hydroxyl termini of linear duplex DNA templates. Here we describe the properties of this unexpected reaction and its potential significance in the context of current models for the structure and catalytic mechanism of the RNA polymerase II active site.
Figure 1:
SIII promotes synthesis
of unanticipated RNA products. A, runoff transcription assays
were performed as described under ``Experimental Procedures''
in the absence or presence of native rat SIII and the indicated
templates. B, SIII-dependent transcription assays were
performed as described under ``Experimental Procedures'' with
50 ng of the indicated template. Where indicated, reactions contained
SIII. Template DNA was omitted from the reactions in lanes 9 and 10; RNA polymerase II was omitted from the reactions
in lanes 13 and 14; and the reactions in lanes 11 and 12 contained 1 µg/ml -amanitin. C,
SIII-dependent transcription assays were performed as described under
``Experimental Procedures'' with
10 ng of the indicated
template and
20 ng (lanes 2 and 7) or 40 ng (lane 3) of native rat SIII or 1 µl (lanes 4 and 8) or 2 µl (lane 5) of recombinant SIII,
renatured as described under ``Experimental Procedures.'' The diagram at the bottom shows the template fragments
used in the experiments shown in this figure and in Fig. 3, Fig. 4, and Fig. 5. E, EcoRI; H, HindIII; N, NdeI; P, PvuII.
Figure 3:
Nuclease sensitivity of SIII-dependent
transcripts. A, E/H1 and E/H2 reaction products. B, gel-purified E/H1 (lanes 1-6) or E/H2 (lanes
7-12) were incubated with 1 unit of DNase I (lanes 2 and 8), 2 units of RNase H (lanes 3 and 9), 0.2 µg/ml RNase A (lanes 5 and 11),
2 µg/ml RNase A (lanes 4 and 10), or a mixture
containing 1 unit of DNase I and 2 µg/ml RNase A (lanes 6 and 12) as described under ``Experimental
Procedures.'' B, promoter-specific runoff transcripts
were synthesized as described under ``Experimental
Procedures'' and digested with 1 unit of DNase I (lane
2), 2 units of RNase H (lane 3), 2 µg/ml RNase A
(10, lane 4), 0.2 µg/ml RNase A (1
, lane
5), or a mixture containing 1 unit of DNase I and 2 µg/ml
RNase A (lane 6).
Figure 4:
SIII-dependent transcription requires a
template with a 3`-hydroxyl terminus. A, promoter-specific
runoff transcription assays using E/N and ddE/ddN DNA templates were
carried out as described under ``Experimental Procedures.''
Reactions in lanes 1, 2, 3, and 4 contained 2, 6, 20, and 60 ng of E/N DNA template. Reactions
in lanes 5, 6, 7, and 8 contained
2, 6, 20, and 60 ng of ddE/ddN DNA template. B,
SIII-dependent transcription assays were carried out as described under
``Experimental Procedures.'' The reactions in lanes 9 and 10 contained 6 ng and 20 ng of E/N DNA template,
respectively. The reactions in lanes 11 and 12 contained 6 ng and 20 ng of ddE/ddN DNA template. To prepare the
ddE/ddN template, 10 µg of pDN-AdML was digested with EcoRI and NdeI. After digestion, 12 units of DNA
polymerase I Klenow fragment (Boehringer Mannheim), ddATP (75
µM final concentration), and ddTTP (75 µM final concentration) were added and incubated at 37 °C for 60
min. The resulting template fragment was purified from a 1.5% low
melting temperature agarose gel as described under ``Experimental
Procedures.''
Figure 5:
The electrophoretic mobility of a
5`-P-labeled DNA template fragment is shifted upon
SIII-dependent transcription. SIII-dependent transcription assays were
carried out as described under ``Experimental Procedures''
except that the reactions in lanes 3 and 4 were
performed in the presence (lane 3) or absence (lane
4) of SIII with
10 ng of
-
P-labeled E/H DNA
and unlabeled ribonucleoside triphosphates. The reaction in lane 1 contained
10 ng of E/H DNA template in the presence of SIII. Lane 2 contains
0.03 ng of
-
P-labeled
E/H DNA as a marker. To prepare the end-labeled DNA template, 5 µg
of E/H DNA fragment was treated with calf intestinal alkaline
phosphatase and incubated with 160 µCi of
[
-
P]ATP in the presence of T4
polynucleotide kinase.
Further investigation revealed that synthesis of SIII-dependent
transcripts on a variety of DNA fragments with or without functional
class II core promoters neither requires nor is stimulated by the
general initiation factors (Fig. 1B, lanes
1-6, Fig. 2, and data not shown). Synthesis of these
transcripts depends strongly, however, on both RNA polymerase II and
template DNA (lanes 7-14). In addition, synthesis of
these transcripts is sensitive to concentrations of -amanitin that
inhibit RNA polymerase II but not mitochondrial RNA polymerase or RNA
polymerases I and III, arguing strongly that the RNA polymerase II
active site is responsible for SIII-dependent transcription (lanes
11 and 12). Finally, we observe that the pattern of
transcripts synthesized in the presence of recombinant SIII and native
rat SIII is similar (Fig. 1C), indicating that the
reaction is due to SIII and not to a contaminant in the native SIII
preparation.
Figure 2:
SIII-dependent RNA synthesis requires DNA
ends. SIII-dependent transcription assays were carried out as described
under ``Experimental Procedures'' in the presence (lanes
1, 3, 5, and 7) or absence (lanes
2, 4, 6, and 8) of SIII with 50 ng
of uncut pUC18 (supercoiled DNA, lanes 1 and 2), KpnI-digested pUC18 (3`-protruding ends, lanes 3 and 4), SalI-digested pUC18 (5`-protruding ends, lanes 5 and 6), or HincII-digested pUC18
(blunt ends, lanes 7 and 8) as
template.
Upon DNase I digestion, the electrophoretic mobility of the E/H1 product in polyacrylamide gels containing 7 M urea decreased; the electrophoretic mobility of the DNase I digestion product was slightly greater than that of the denatured tem-plate fragment. Because the electrophoretic mobility of double-stranded nucleic acid is greater than that of single-stranded nucleic acid, this result suggests that the E/H1 product has an unusually stable secondary structure that is largely resistant to 7 M urea. Consistent with this possibility, E/H1 was susceptible to digestion by RNase H and by a mixture of DNase I and RNase A, but was largely resistant to digestion with RNase A. Taken together, these results suggest that E/H1 may be a double-stranded, intramolecular ``snapback'' hybrid of the SIII-dependent transcript and its DNA template; such a product would be synthesized if polymerase initiates its synthesis by extending the 3`-OH of one strand of the DNA molecule and then uses that same DNA strand as template. Regardless of the exact structure of the E/H1 product, our observation that its RNA portion is sensitive to RNase H and, therefore, exists as a DNA-RNA hybrid, argues strongly that synthesis of the E/H1 product is template-directed.
The electrophoretic mobility of the E/H2 product increased upon DNase I digestion. In contrast to the E/H1 product, the E/H2 product was largely resistant to both RNase A and RNase H, suggesting that this SIII-dependent RNA product is capable of forming a double-stranded RNA-RNA hybrid; however, the precise structure of this product is unclear.
As a control for the purity and specificity of the nucleases used in these experiments, the single-stranded RNA products of promoter-specific transcription reactions were digested with the same enzyme preparations used to analyze the E/H1 and E/H2 products. As expected, promoter-specific runoff transcripts synthesized from the AdML promoter were resistant to digestion by DNase I and RNase H, but were sensitive to digestion by RNase A (Fig. 3C).
As a control, we compared the abilities of the E/N fragment and the dideoxynucleotide-treated E/N fragment (ddE/ddN) to support promoter-specific transcription from the AdML promoter in the presence of RNA polymerase II, TBP, TFIIB, TFIIE, TFIIF, and TFIIH. As shown in Fig. 4, lanes 1-8, both the E/N and ddE/ddN fragments supported synthesis of runoff transcripts from the AdML promoter. When transcription was carried out in the presence of just RNA polymerase II and SIII, however, the ddE/ddN fragment failed to support efficient SIII-dependent transcription (Fig. 4, compare lanes 9-11 with lanes 12-14).
It is well established that purified RNA polymerase II is
capable of initiating transcription accurately at the
promoter-regulatory regions of eukaryotic protein-coding genes in the
presence of a set of general initiation factors referred to as TFIID,
TFIIB, TFIIE, TFIIF, and TFIIH(8) . In the absence of these
initiation factors, RNA polymerase II does not initiate at promoters,
but is capable of initiating transcription de novo on
single-stranded or supercoiled DNA templates and at nicks or ends of
duplex DNA(12, 25, 26) . In addition, RNA
polymerase II has been shown to catalyze template-directed extension of
free DNA 3`-hydroxyl termini at sites of single strand nicks in duplex
DNA(27, 28, 29) . Lewis and Burgess (27) have shown that extension of 3`-hydroxyl termini at
single-strand breaks in duplex DNA is the predominant reaction when
transcription is carried out in the presence of Mg,
whereas de novo initiations predominate at these sites when
transcription is carried out in the presence of Mn
.
In this report, we have shown that general elongation factor SIII
possesses the novel ability to promote template-directed extension by
RNA polymerase II of 3`-hydroxyl termini of duplex DNA fragments. This
reaction depends strongly on the presence of a free 3`-hydroxyl
terminus, but does not exhibit a strong preference for 3`-protruding,
3`-recessed, or blunt DNA ends. Interestingly, SIII appears to have a
negligible effect on de novo synthesis of end-to-end
transcripts initiated by RNA polymerase II on the same DNA fragments
(data not shown). Under the conditions used in our assays (7
mM MgCl
, 28 °C), de novo initiation
at the ends of DNA templates and synthesis of end-to-end transcripts is
barely detectable in either the absence or presence of SIII.
What is the relationship between SIII's ability to promote template-directed extension of DNA primers by RNA polymerase II and its ability to suppress pausing by polymerase during RNA chain elongation? It is unlikely that SIII-dependent extension of duplex DNA ends by RNA polymerase II is a physiologically important reaction, because evidence that it occurs in vivo is lacking. As suggested by Salzman and co-workers(29) , the template-directed addition of ribonucleotides to 3`-hydroxyl termini of DNA by RNA polymerase II may occur in a reaction that mimics formation of the RNA polymerase II ternary elongation complex. In this case, RNA polymerase II would bind the 3`-hydroxyl terminus of DNA in its active site, just as the enzyme binds the 3`-end of an elongating RNA molecule; the polymerase catalytic site for ribonucleotide addition would then use the DNA 3`-hydroxyl terminus as if it were the 3`-hydroxyl terminus of the nascent RNA transcript. In light of this model, our observation that SIII promotes extension by RNA polymerase II of the 3`-hydroxyl termini of DNA primers is consistent with the idea that SIII may facilitate proper positioning of DNA 3`-hydroxyl termini (and, by extension, the 3`-hydroxyl termini of nascent transcripts) with respect to the polymerase catalytic site. If this is correct, the mechanisms by which SIII promotes extension of DNA 3`-ends and suppresses transient pausing by the RNA polymerase II elongation complex could be closely related.
Elongation by RNA polymerase II is an inherently discontinuous process, often punctuated by transient pauses at many sites along DNA templates or by transcriptional arrest at specific DNA sequences referred to as intrinsic arrest sites(1, 30) . It has been suggested that transcriptional arrest occurs when the polymerase catalytic site for nucleotide addition slips more than 7 or 8 nucleotides upstream from the 3`-hydroxyl terminus of the nascent transcript(31, 32) . The arrested elongation complex is inactive until released from arrest by the action of elongation factor SII, which promotes cleavage of the nascent transcript upstream of its 3`-terminus, thereby creating a new RNA 3`-terminus that is correctly positioned with respect to the catalytic site(3) . If transient pausing results when the polymerase catalytic site is only slightly displaced from its position at the 3`-end of growing transcripts, it is possible that SIII promotes efficient RNA chain elongation by ensuring that the catalytic site and 3`-hydroxyl terminus of the nascent transcript are kept in register.