From the Department of Microbiology and Immunology, Morse Institute for Molecular Genetics, State University of New York, Health Science Center, Brooklyn, New York 11203-2098
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ABSTRACT |
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T7 RNA polymerase (RNAP) is able to traverse a variety of discontinuities in the template (T) strand of duplex DNA, including nicks, gaps, and branched junctions in which the 3' end of the T strand is not complementary to the non-template (NT) strand. The products represent a faithful copy of the T strand, with no insertions or deletions. On double-stranded templates having protruding 3' ends the polymerase is able to insert the free 3' end of the NT strand and to utilize this as a new T strand ("turn around transcription"), resulting in the anomalous production of high molecular weight transcripts.
The capacity of T7 RNAP to bypass interruptions in the T strand depends upon the stability of the elongation complex. Sequences that are expected to stabilize a local RNA:DNA hybrid (such as the presence of a C6 tract in the T strand) dramatically reduce dissociation of the RNAP while still allowing the enzyme to insert a new 3' end. Similar effects on RNAP release are observed when the enzyme reaches the end of a template (i.e. when synthesizing runoff products), resulting in markedly different yields of RNA product during multiple rounds of transcription.
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INTRODUCTION |
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T7 RNA polymerase (RNAP)1 is a single subunit enzyme that is able to carry out all of the steps in the transcription process without the need for auxiliary factors (for review see Ref. 1). The crystal structure of the enzyme has been solved to a resolution of 3.3 Å (2), and it is therefore highly attractive as a model for studies of the mechanism of RNA synthesis (for review see Refs. 3 and 4).
During the early stages of transcription, T7 RNAP engages in multiple cycles of initiation in which short RNA products are synthesized and released without movement of the RNAP away from the promoter (5-9). Once the enzyme has initiated transcription and cleared the promoter, the ternary elongation complex is highly processive and is able to synthesize long RNA chains (>15,000 nt) until it encounters a termination signal or reaches the end of the template. The transition from an unstable initiation complex to a stable elongation complex (EC) is accompanied by isomerization of the complex (6, 10, 11) and may involve wrapping of a flexible "thumb" domain (12, 13) and possibly a "specificity loop" (1, 14) around one or both strands of the template, leading to partial closure of the DNA binding cleft.
The non-template (NT) strand of the DNA does not appear to be required
for initiation or for the early stages of transcription, as Maslak and
Martin (15) have shown that T7 RNAP can initiate transcription at a
promoter that is single-stranded in the initiation region (from 6 and
downstream), and Milligan et al. (7) have shown that the
enzyme can transcribe a single-stranded DNA template once it has
initiated transcription from a double-stranded promoter. However, Zhou
et al. (16, 17) have suggested that the stability of the EC
may require the presence of duplex DNA at the leading edge of the
elongation complex, as an elongation complex that is halted just
upstream from a region in which the NT strand is missing appears to be
unable to resume transcription.
A number of studies to determine the importance of the integrity of the template (T) strand to processive transcription have been carried out. Although there are conflicting reports in the literature with regard to the ability of T7 RNAP to bypass nicks in the T strand (18, 19), Zhou et al. (16, 17) observed that T7 RNAP is able to bypass gaps of 1-24 nt, resulting in a faithful copy of the T strand in the regions preceding and following the gap. Although this result demonstrated that the enzyme is able to thread the 3' end of a paired T strand into the active site, the RNAP was unable to switch to a free oligomer of single-stranded DNA, leading these authors to conclude that the DNA template must be double-stranded at the leading edge of the EC in order for template switching to occur (17). However, an alternate explanation for this observation is that if the downstream oligomer is not tethered in the vicinity of the EC (because it is not complementary to the NT strand) its local concentration is low, and switching to the new T strand is inefficient.
In this work, we have explored the ability of T7 RNAP to traverse nicks and gaps in the template strand and also to jump across branched junctions in which the leading or trailing edges of the T strand are not complementary to the NT strand. We found that the RNAP is able to traverse all of these discontinuities. Our results do not support a requirement that the DNA must be double-stranded at either the leading or trailing edge of the elongation complex in order for strand switching to occur but suggest an important role for the NT strand in tethering the T strand in the vicinity of the EC.
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EXPERIMENTAL PROCEDURES |
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DNA Templates and RNA Polymerase--
DNA oligomers were
synthesized by Macromolecular Resources (Colorado State University) and
purified by low pressure reverse phase chromatography. To prepare
synthetic templates, the indicated combinations of oligomers were mixed
together (final concentration 0.5 µM, each oligomer) in
40 µl of GHT buffer (30 mM K-HEPES, pH 7.8, 100 mM potassium glutamate, 15 mM
Mg(OAc)2, 0.25 mM EDTA, 0.05% Tween 20 (20)),
and the samples were heated to 70 °C for 10 min and cooled slowly to
room temperature (2-3 h). The templates were either used immediately
or stored at 20 °C.
Transcription Assays--
Unless otherwise noted, transcription
was carried out in a volume of 10 µl in GHT buffer (see above)
containing 0.5 mM ATP, CTP, GTP, and UTP (Amersham
Pharmacia Biotech Ultrapure); 2 µCi of [-32P]ATP or
GTP (specific activity of 800 Ci/mmol; NEN Life Science Products);
10-20 nM RNA polymerase, and 50 nM synthetic
DNA or 1 µg of plasmid DNA as template. Reactions in which ITP
replaced GTP contained 0.5 mM ITP and 2 mM GMP
to allow efficient initiation (23). Reactions were incubated at
37 °C for 10 min and terminated by the addition of 10 µl of stop
buffer, and the products were resolved by electrophoresis in
polyacrylamide gels containing 7 M urea (21). The
radioactivity in each electrophoretic species was quantified by
exposing the gel to a PhosphorImagerTM screen (Molecular
Dynamics) using a Storm 860 scanner and ImageQuaNT version 4.2a
software (Molecular Dynamics). The efficiency of gap or branch jumping
was calculated as follows: efficiency = (long runoff
product)/(short runoff product + long runoff product) taking into
account the base composition of individual transcripts.
RNA Sequence Determination--
Transcription reactions were
carried out as described above except that the volume was increased to
100 µl, and 10 units of RNase inhibitor (Boehringer Mannheim) were
added. After 1 h at 37 °C, samples were treated with 1 unit of
DNase (RNase-free, Promega) for 20 min and extracted with phenol and
chloroform. RNA was precipitated at 70 °C in the presence of 70%
isopropyl alcohol and 0.3 M sodium acetate and resolved by
electrophoresis in a 20% polyacrylamide gel as described above. Gel
slices containing the RNA were excised, crushed in 500 µl of elution
buffer (10% sodium acetate in TE (0.01 M Tris-HCl, pH 7.4;
0.1 mM EDTA)), and incubated overnight at 37 °C. The
sample was extracted with phenol and chloroform, and the RNA was
precipitated with isopropyl alcohol and taken up in 10 µl of TE. A
cDNA copy of the RNA was synthesized using Moloney murine leukemia
virus reverse transcriptase and a MarathonTM cDNA
amplification kit (CLONTECH); the primer for first
strand synthesis was 5'-CAGGATCCCTCTAGAACTAGTGG-3', which
contains a BamHI site (underlined). The cDNA was
amplified in a polymerase chain reaction using a
PrimeZymeTM kit (Biometra), the primer noted above, and a
second primer (5'-CAGAATTCGGGAGACCACAACCTC-3') that
contains an EcoRI site (underlined). The resulting products were digested with EcoRI and BamHI and cloned
into the corresponding sites of pBluescript II SK(+) (Stratagene); the
DNA sequence of the cloned interval was determined using chain
terminating dNTPs (22).
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RESULTS |
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T7 RNAP Is Able to Bypass Nicks and Gaps in the Template Strand-- To examine the effects of discontinuities in the DNA template on processivity, we constructed double-stranded templates having no discontinuities, nicks, gaps of 1-2 nt, or branches in the T strand by annealing synthetic T and NT strand DNA oligomers (Fig. 1A, see Table I for sequences). These templates were then transcribed by T7 RNAP, and the products were resolved by electrophoresis in 20% polyacrylamide gels (Fig. 1, B and C).
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T7 RNAP Can Transcribe Branched Templates-- The ability of the polymerase to jump across a branch in the template (in which the 3' end of the T strand at the distal edge of the discontinuity is not complementary to the NT strand) was examined as shown in Fig. 1C. In this experiment, T strand oligomers having unpaired 3' tails 4-16 nt long were positioned either immediately after the 5' end of the upstream T strand oligomer (lanes 9, 11, 13, 15, and 17) or after a gap of 1 nt (lanes 10, 12, 14, 16, and 18). The polymerase is able to transcribe through all of these junctions, resulting in the synthesis of transcripts that are larger than the product from a fully double-stranded template (lane 1, 47 nt) by an increment that corresponds to the length of the branch minus the size of the gap (if present). The efficiency of branch jumping drops with increasing length of the branch, but a plot of the data (Fig. 1D) suggests that a basal level of branch jumping may remain even with longer branches. Further experiments with templates having longer branches will be required to confirm this.
The products arising from transcription across the branch junctions were discrete and homogeneous and of a size expected for an exact copy of the branched T strand. The accuracy of transcription across such a junction was confirmed by sequencing a cDNA copy of the "jump" transcript produced from template 11, which has a branch of 8 nt. The results demonstrate that the transcript represents an accurate copy of the T strand on either side of the discontinuity and that T7 RNAP does not insert or delete nucleotides when it traverses such a junction (Fig. 2).
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Anomalous Transcription of Templates with Protruding 3' Ends Is Due to Branch Jumping-- T7 RNA polymerase is known to synthesize anomalous, high molecular weight products from templates that have protruding 3' ends (24). This phenomenon depends upon the structure at the termini, but not the sequence (24-26), and is not due to initiation at the ends of the DNA, as it requires the presence of a promoter in the template (24, 26). In view of the finding that the RNAP is able to insert a free 3' end when it reaches the end of a T strand, we asked whether the synthesis of these unusual products might arise from the same phenomenon (Fig. 3). Although transcription of templates that have a blunt end results in the synthesis of the expected runoff products (lanes 1 and 2), transcription of templates that have a protruding 3' end gives rise to additional RNAs that would result from insertion of the 3' end of the NT strand and subsequent transcription of that strand (lanes 3-5). If the terminus of the template upstream from the promoter has a blunt or recessed 3' end, the process ceases there (lanes 4 and 5, 71 nt). However, if the upstream terminus has a protruding 3' end (lane 3) the polymerase can again insert the free end, re-transcribing the template strand. Repeated cycles of transcription around the DNA by this mechanism ("turn around transcription") result in the synthesis of a ladder of products and the accumulation of high molecular weight RNAs that do not enter the gel. As noted elsewhere, the production of these spurious products can be avoided through the use of T7 RNAP mutants such as del172-3 (25, 26).
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Effects of G:C Tracts on Branch Jumping and Runoff Transcription-- In the experiment described in Fig. 1, a C6 tract was included in the T strand to enhance the formation of a stable DNA duplex in the vicinity of the discontinuity when paired with a complementary G6 tract in the NT strand. To determine how the presence of this G:C "clamp" affects transcription, we positioned the G:C tract either before or after the discontinuity (Fig. 4). In a completely duplex DNA template (one having no discontinuities in the T strand) only full-length runoff products (47 nt) were synthesized, with no evidence of pausing near or within the G:C tract (Fig. 4B, lane 1). When the G:C tract was placed downstream from the discontinuity (as in Fig. 1) the characteristic production of short RNAs (24 nt) that terminate at the proximal side of the discontinuity was observed, along with production of the jump products (47 or 51 nt; Fig. 4B, lanes 2 and 3). In contrast, when the G:C clamp was placed prior to the discontinuity, production of the short RNA was not detected, whereas synthesis of full-length runoff (jump) products continued to be observed (Fig. 4B, lanes 4 and 5). Under the latter conditions, the apparent efficiency of branch and gap jumping approaches 100%, as the jump transcripts are practically the only products observed.
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Effects of Alternate Structures at the DNA Terminus on Product Yield-- In view of the observations above, we explored the effects of other variations in structure and sequence at the ends of a DNA template on the yield of RNA products (Fig. 6). In these experiments, the activity of templates that ended in various homopolymeric tracts was compared with that of a control template that ended with the sequence 5'-GACTAC-3' (in the NT strand), either when the T strand was complementary to the NT strand or was unpaired.
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DISCUSSION |
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We have shown that T7 RNAP is able to bypass a variety of interruptions in the template strand and that the sequence at the end of the strand affects the stability of the complex and its ability to traverse the discontinuity. Our observation that the enzyme can cross gaps of 1-2 nt in the T strand is in agreement with a previous report demonstrating jumping of gaps up to 24 nt in length (17). However, in that report, the authors concluded that template switching by T7 RNAP occurred only if the 3' end of the T strand was complementary to the NT strand (i.e. was present in the form of a DNA duplex). In this work, we have shown that T7 RNAP may also insert the 3' end of an unpaired T strand when it is located in a branch junction. Under these circumstances, the 3' end of the T strand is present at a high local concentration (as it is tethered to the EC via its complementarity to the downstream region of the NT strand), and this may account for failure on the part of previous investigators to detect jumping of T7 RNAP to a second molecule added in trans. We have also found that T7 RNAP is able to traverse branch junctions in which the T strand is unpaired before the discontinuity (i.e. has a 5' tail) or in which there are unpaired regions on both sides of the discontinuity (having both 3' and 5' tails), although with reduced efficiency in the latter case (30). In contrast to these results, Nudler et al. (28) have reported that Escherichia coli RNAP may also switch to a new template strand requirement for double-stranded DNA for template switching by T7 RNAP suggests that there may be important structural and functional differences between the bacterial and phage enzymes.
There are conflicting observations regarding the importance of the NT strand to the processivity and stability of the T7 RNAP elongation complex. Some experimental results suggest that the polymerase sequesters primarily the T strand and that a continuous encirclement of both strands may not be required for processivity. For example, polymerases that approach one another from opposite directions over the same transcription region are able to pass one another with no apparent pausing or termination, and we have observed processive transcription on single-stranded templates over distances as great as 800 nt (29).2 Furthermore, cross-linking of psoralen mono-adducts or of psoralen-derivatized oligonucleotides inhibits transcription by T7 RNAP when the cross-link is to the T strand but not to the NT strand (31, 32), and we have observed similar results in the case of a protein cross-linked to the T or NT strands.3
Although these results indicate that the presence of the NT strand is not required for the stability (and hence processivity) of an actively transcribing complex, Zhou et al. (17) reported that an elongation complex that is halted just upstream from a region in which the NT strand is missing is unable to resume transcription when the remaining substrates are added and suggested that the NT strand may be required for the stability of such a complex. In work to be reported elsewhere, we have found differences in the stabilities and properties of halted complexes versus actively transcribing complexes, and we have shown that the presence of the NT strand is more important to the stability of the former complexes than to the latter.4,5
The effects of various configurations and sequences at the ends of the DNA template on product yield and enzyme release indicate that the stability of an RNA:DNA hybrid is important in maintaining the association of the ternary complex and that under certain circumstances product release and complex dissociation may be rate-limiting. Thus, for example, we have found that a terminal C6 tract in the T strand inhibits dissociation and that this effect may be relieved by substituting ITP for GTP in the reaction. We have observed similar effects on templates that end in a variety of G:C-rich sequence contexts, indicating that it is the stability of the RNA:DNA hybrid, and not its sequence, that causes this effect (30).
Due to the particularly low stability of rU:dA base pairs (27), the
premature dissociation of complexes observed at the end of a template
having a terminal A6 tract in the T strand (Fig. 6) is
expected. However, the lower yield of transcripts from such a template
is not consistent with a lowered hybrid stability (which should
increase the turnover rate and hence product yield). As noted above, a
possible explanation for this phenomenon is that the low stability of
rU:dA base pairs may result in a decreased rate of elongation, thus
increasing the time required to synthesize an rU:dA hybrid long enough
to destabilize the complex and promote dissociation. In exploring this
phenomenon, we examined the effects of a longer dA tract at the end of
the T strand (an A10 tract) and found that very few
polymerases were able to reach the end of this template (Fig. 6,
lane 11). Under these conditions the longest products
synthesized appeared to have incorporated only 8 U residues, suggesting
that this is the maximum length of an extended rU:dA hybrid that the
enzyme can tolerate when approaching the end of the DNA or that the
enzyme slips to the end of the template after the incorporation of 7-8
consecutive UMP residues (33). In previous work, we examined the
effects of extended poly(dA) tracts in the T strand when these are
embedded within a transcription unit (i.e. are not located
at the end of the template) and found that they destabilize T7 RNAP,
sometimes causing termination but also resulting in slippage of the
transcription complex on the template (33). These effects were greatly
enhanced at lower concentrations of UTP, and under these conditions the
slippage products were observed to approach a limit size that would
correspond to the incorporation of 8-10 consecutive UMP residues (33). These observations may have relevance to the process of termination. Class I termination signals for T7 RNAP (e.g. T) as well
as rho-independent termination signals for E. coli RNAP
encode RNAs with a stable stem-loop structure followed by a run of
uridylate residues. It seems likely that these U runs function both to
destabilize the transcription complex as well as to slow the passage of
the enzyme through the signal (34).
The picture that emerges from these and other studies of T7 RNAP is of a transcription complex that is held together by dynamic interactions between the RNAP, the DNA template, and the RNA product. As the enzyme approaches the end of the template strand, potential contacts with downstream regions of the template are lost, resulting in decreased stability of the complex.4 At this point, the stability of the RNA:DNA hybrid becomes critical to the continued maintenance of the complex. If the strength of the hybrid is high (e.g. in a G:C-rich sequence context) the complex is slow to dissociate, allowing the enzyme to position a new T strand 3' end (if available) in the active site and to resume transcription. If the strength of the hybrid is low (e.g. in an rU:dA context), dissociation is more likely to occur before the enzyme can resume transcription.
Under normal conditions of transcription in which the enzyme is highly processive (i.e. the presence of an intact duplex DNA template downstream, the absence of a pause or termination signal, and non-limiting concentrations of substrate), changes in the strength of the local RNA:DNA hybrid generally do not decrease the stability of the EC below the threshold that would result in dissociation. Thus, the RNAP does not usually terminate in extended T:A-rich regions nor does it pause in G:C-rich tracts (see Ref. 33 and this work). The contribution of the RNA:DNA hybrid to complex stability is likely to become of particular importance when the EC is destabilized or is halted for some reason (e.g. upon reaching the end of the template, or in the context of a pause/termination signal (35)2). In work to be reported elsewhere, we have found that the structure and stability of the T7 transcription complex is dynamically determined and that the properties of a halted EC are quite different from an EC that is involved in active transcription.4,5 Efficient release of the complex at the ends of the template is stimulated in the presence of T7 lysozyme (which enhances recognition of certain types of termination signals (26, 35)) or by the use of T7 RNAP mutants with altered termination properties.6
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ACKNOWLEDGEMENTS |
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We are grateful to Ray Castagna for technical assistance and to Rita Gould and Roseann Lingeza for secretarial assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM38147 (to W. T. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Submitted this work in partial fulfillment of the requirements for
a doctoral degree at the State University of New York.
§ Present address: Dept. of Hematology and Oncology, Wexner Pediatric Research Institute, Children's Hospital, 700 Children's Dr., Columbus, OH 43221.
¶ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Morse Institute for Molecular Genetics, State University of New York, Health Science Center at Brooklyn, 450 Clarkson Ave., Box 44, Brooklyn, NY 11203-2098. Tel.: 718-270-1238; Fax: 718-270-2656; E-mail: pogo51{at}aol.com.
1 The abbreviations used are: RNAP, RNA polymerase; nt, nucleotide(s); EC, elongation complex; NT, non-template; T, template.
2 B. He, A. Kukarin, D. Temiakov, S. T. Chin-Bow, D. L. Lyakhov, M. Rong, R. K. Durbin, and W. T. McAllister, submitted for publication.
3 R. K. Durbin and W. T. McAllister, unpublished observations.
4 V. Gopal, W. T. McAllister, and R. Sousa, submitted for publication.
5 P. E. Karasavas, S. T. Chin-Bow, R. Sousa, and W. T. McAllister, submitted for publication.
6 M. R. Rong, A. Kukarin, and W. T. McAllister, manuscript in preparation.
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REFERENCES |
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