(Received for publication, July 10, 1995; and in revised form, August 1, 1995)
From the
We examine the association of transcription factor TFIIIA with RNA-DNA heteroduplexes containing sequences from the Xenopus borealis 5 S rRNA gene. Under conditions where TFIIIA selectively binds to 5 S rRNA or the internal control region of the 5 S rRNA gene, no specific association of TFIIIA with DNA-RNA heteroduplexes containing either strand of 5 S DNA could be detected. We discuss our results with respect to specific models of TFIIIA recognition of the internal control region and of DNA-RNA hybrids by zinc finger proteins.
TFIIIA is a positive transcription factor for the 5 S ribosomal
RNA gene (Engelke et al., 1980; Pelham and Brown, 1980). The
protein has a modular structure comprised of nine zinc finger domains
(Hanas et al., 1983; Smith et al., 1984; Ginsberg et al., 1984; Miller et al., 1985) that are linked to
a carboxyl-terminal domain that interacts with other transcription
factors (Hayes et al., 1989; Mao and Darby, 1993). The nine
zinc fingers bind as a linear array along the internal control region
(ICR) ()of the 5 S rRNA gene (Smith et al., 1984;
Vrana et al., 1988). The exact mode of interaction of the zinc
fingers with the ICR has been controversial (Fairall et al.,
1992; Fairall and Rhodes, 1992). A feature of early models was the
suggestion that A-type DNA could be involved in TFIIIA binding (Rhodes
and Klug, 1986; Fairall et al., 1989). Other experiments
suggested that TFIIIA recognizes B-type DNA (Gottesfeld et
al., 1987; Hayes et al., 1990). In addition, the path of
DNA is known to be distorted through interaction with TFIIIA (Schroth et al., 1989; Bazett-Jones and Brown, 1989). The solution of
the crystal structure of the zinc finger protein Zif 268 indicated that
zinc finger proteins of the TFIIIA class can interact with a B-type
helix (Pavletich and Pabo, 1991; Nekludova and Pabo, 1994). However,
the contacts made with the double helix by Zif 268 are predominantly to
a single strand of the helix. It has recently been suggested that a
combination of an A-type DNA structure and predominant recognition of a
single strand of the double helix might allow TFIIIA to bind in a
sequence-selective manner to a DNA-RNA heteroduplex (Shi and Berg,
1995).
Gottesfeld and colleagues have clearly established that distinct clusters of zinc fingers within TFIIIA have different affinities for DNA sequences compared with RNA sequences (Liao et al., 1992; Clemens et al., 1993, 1994; McBryant et al., 1995). These studies indicate that TFIIIA utilizes distinct domains to confer specificity to the interaction with 5 S DNA and 5 S RNA; however, all zinc finger domains contribute to the stability of association with nucleic acid. Other experiments that examine the interaction of TFIIIA with 5 S rRNA are consistent with the protein recognizing RNA through different mechanisms than the recognition of DNA (Darby and Joho, 1992; Theunissen et al., 1992; Romaniuk et al., 1987; You et al., 1991; Sands and Bogenhagen, 1987, 1991). Since TFIIIA can recognize both DNA and RNA with sequence selectivity it is possible that TFIIIA might recognize RNA-DNA heteroduplexes with comparable sequence selectivity.
In this work we directly examine the interaction of TFIIIA with DNA-RNA heteroduplexes containing either strand of the 5 S rRNA gene as DNA. In contrast with earlier speculations (Rhodes and Klug, 1986; Fairall et al., 1989; Shi and Berg, 1995), we find that TFIIIA does not have sequence-selective interactions with these specific RNA-DNA heteroduplexes under the same conditions that it selectively binds to 5 S rRNA or the 5 S rRNA gene.
Figure 1: Preparation of ``sense'' or ``antisense'' RNA-DNA heteroduplexes containing the Xenopus 5 S rRNA gene sequence. The scheme illustrates the organization of the plasmid pXP10, where the arrowedregion represents the 5 S rRNA gene and the hatchedarea the ICR to which TFIIIA binds. The alternative construct used shows pXP14 contains the same 5 S rRNA sequence, but it is inverted (Wolffe et al., 1986). The arrow at 1 indicates the start of the SP6 polymerase transcript.
Figure 2: Gel retardation assays showing TFIIIA binding to both DNA-RNA heteroduplexes and to DNA and 5 S rRNA. A, DNA and 5 S rRNA. TFIIIA binding to a 298-bp fragment of the Xenopus 5 S rRNA gene (DNA, lanes 1-6) or to 5 S rRNA (RNA, lanes 7-12) is shown. 5 ng of end-labeled nucleic acid (lanes1 and 7) was incubated with 0.15 ng (lanes2 and 8), 0.3 ng (lanes3 and 9), 0.6 ng (lanes4 and 10), 1.2 ng (lanes5 and 11), and 2.4 ng (lanes6 and 12) of TFIIIA. Complexes were resolved on a 0.7% agarose gel. The resolution of free nucleic acid (Free) and the binding of single (1) or multiple TFIIIA molecules (2, 3) to the fragments is indicated, as are the generation of nucleoprotein aggregates. B, DNA-RNA heteroduplex. 5 ng of ``sense'' (XP14) (lanes1-5) or ``antisense'' (XP10) (lanes 6-10) RNA-DNA heteroduplex (lanes1 and 6) was incubated with 15 ng (lanes2 and 7), 30 ng (lanes3 and 8), 60 ng (lanes4 and 9), and 120 ng (lanes5 and 10) of TFIIIA.
Figure 5: Footprinting analysis of TFIIIA binding to both DNA-RNA heteroduplexes and to DNA. A, TFIIIA binding to 5` end-labeled ``sense'' (XP14) and ``antisense'' (XP10) heteroduplexes was analyzed by DNase I digestion as described under ``Materials and Methods.'' The lanes are labeled as follows: lanes2 and 6, DNase I digestion of free heteroduplex; lanes3 and 4 or 7 and 8, digestion of heteroduplex with one and two TFIIIA molecules bound to the fragment, respectively. Lanes1 and 2 show labeled G cleared fragments in a Maxam-Gilbert G ladder. B, TFIIIA binding to double-stranded DNA of the same sequence present in the heteroduplexes. The lanes are labeled as follows: -, DNase I digestion of free DNA (lanes10 and 13); +, DNase I digestion of the same fragment bound by a single TFIIIA molecule (lanes11 and 14). Lanes9 and 12 are of labeled G cleared fragments in a Maxam-Gilbert G ladder. The position of the ICR is indicated.
Figure 3:
Binding titration for TFIIIA binding to
either ``antisense'' () or ``sense'' DNA-RNA
heteroduplexes (⊡). Autoradiographs of gel mobility shifts were
scanned with a laser densitometer, and the fraction of bound nucleic
acid was plotted against the TFIIIA concentration used. A titration
curve of TFIIIA binding to DNA is included for comparison
(
).
Figure 4: Specificity and strength of TFIIIA binding to DNA-RNA heteroduplexes. A, TFIIIA binding to either a fragment of the 5 S rRNA gene (DNA, lanes 9-16) or 5 S rRNA (5S rRNA, lanes 1-8). The complexes formed by mixing 5 ng of nucleic acid and 3 ng of TFIIIA (lanes 2-10) were incubated with 10-fold (lanes 3, 6, 11, and 14), 100-fold (lanes 4, 7, 12, and 15), and 500-fold excesses (lanes 5, 8, 13, and 16) of either specific (Sp. containing the 5 S rRNA sequence) or nonspecific DNA (NSp. pBR322) and separated on a 0.7% agarose gel. The resolution of free nucleic acid (Free) and the binding of single (1) or multiple TFIIIA molecules to the fragments is indicated. B, 5 ng of DNA-RNA heteroduplex (``antisense'') (lane1) was incubated with TFIIIA (120 ng) to generate a mixed population of TFIIIA-heteroduplex complexes (lane2). Binding was then assessed upon titration of specific (Sp.) or nonspecific competitor DNA (NSp.) such that the samples contained a 1-fold (lanes3 and 7), 10-fold (lanes4 and 8), 100-fold (lanes5 and 9), or 500-fold excess (lanes6 and 10). The resultant nucleoprotein complexes were separated on a 0.7% agarose gel. C shows the mobility of naked nucleic acid, and T shows the mobility of the complex of TFIIIA with nucleic acid without competitor.
Our final assessment of TFIIIA interaction with the RNA-DNA heteroduplexes was to examine whether sequence-selective binding occurred by DNase I footprinting. Our strategy was to carry out the DNase I cleavage reaction after mixing TFIIIA either with the RNA-DNA heteroduplex or with DNA as a control before resolving the nucleoprotein complexes on a non-denaturing gel (Hayes and Wolffe, 1992). The nucleoprotein complex of interest was then eluted from the gel, and the radiolabeled nucleic acid was isolated and resolved on a denaturing polyacrylamide gel to reveal any footprint. Resolution of the first complex formed when a single molecule of TFIIIA is bound to 5 S DNA reveals a clear footprint over the ICR (Fig. 5B, lanes 9-14). Identical analysis of both DNA-RNA heteroduplexes does not reveal a footprint (Fig. 5A, lanes 1-8). Thus although TFIIIA will bind to either heteroduplex, this interaction is not sequence selective.
The early analysis of TFIIIA interactions with the 5 S RNA gene suggested that TFIIIA might bind preferentially in a sequence-specific manner to the non-coding DNA strand (Sakonju and Brown, 1982). Subsequent studies have clearly established that such selective interactions can occur for both TFIIIA and other zinc finger proteins within the context of double helical DNA (Fairall et al., 1986; Pavletich and Pabo, 1991). The suggestion that comparable strand- and sequence-specific interactions detected in the binding of zinc finger proteins to DNA-RNA heteroduplexes (Shi and Berg, 1995) might also occur with TFIIIA is not supported by our results (Fig. 3Fig. 4Fig. 5). We find that TFIIIA will associate with a DNA-RNA heteroduplex (Fig. 2) but that this interaction is relatively weak and nonspecific compared with binding to 5 S DNA or RNA (Fig. 3Fig. 4Fig. 5) (Clemens et al., 1993).
The lack of sequence-specific recognition of heteroduplexes containing either strand of 5 S DNA is consistent with previous data indicating that TFIIIA recognizes the internal control region as B-type DNA (Gottesfeld et al., 1987; Hayes et al., 1990). Since DNA-RNA heteroduplexes approximate to an A-type conformation (Hall, 1993), our results provide yet further evidence against the proposal that equivalent recognition of DNA and RNA might be mediated by the same zinc fingers within TFIIIA (Rhodes and Klug, 1986; Fairall et al., 1986, 1989). Our results are entirely consistent with the definition of distinct domains of TFIIIA that interact with DNA or RNA in a sequence-selective manner (see Clemens et al.(1993), Darby and Joho(1992), and Theunissen et al.(1992)).
Proteins such as the histones will not interact stably with RNA-DNA heteroduplexes (Dunn and Griffith, 1980; Hovatter and Martinson, 1987). Our results suggest that the interaction of TFIIIA with the 5 S RNA gene will be destabilized by the generation of any RNA-DNA heteroduplex during transcription. Thus the generation of RNA-DNA heteroduplexes might provide a means of destabilizing nucleoprotein complexes within the chromosome. It should, however, be noted that in our experiments we generate very long heteroduplexes (298 bp) whereas in vivo any heteroduplex formed during transcription would be much shorter. TFIIIA in isolation is displaced from DNA by transit of bacteriophage RNA polymerase (Campbell and Setzer, 1991) yet remains bound to DNA within the intact transcription complex (Bogenhagen et al., 1982; Wolffe et al., 1986). Presumably multiple contacts between TFIIIA and other transcription factors that bind outside of the transcribed region facilitate the retention of TFIIIA on the 5 S rRNA gene during the transcription process (Wolffe and Morse, 1990; Kassavetis et al., 1990).