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, the
Laboratory for Molecular Genetics of Microorganisms, Institute
of Molecular Genetics, Russian Academy of Sciences,
46 Kurchatov Square, Moscow 123182, Russia, and the ** V.A.
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences,
32 Vavilov St., Moscow 117984, Russia
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ABSTRACT |
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We have characterized an unusual type of termination signal for T7 RNA polymerase that requires a conserved 7-base pair sequence in the DNA (ATCTGTT in the non-template strand). Each of the nucleotides within this sequence is critical for function, as any substitutions abolish termination. The primary site of termination occurs 7 nucleotides downstream from this sequence but is context-independent (that is, the sequence around the site of termination, and in particular the nucleotide at the site of termination, need not be conserved). Termination requires the presence of the conserved sequence and its complement in duplex DNA and is abolished or diminished if the signal is placed downstream of regions in which the non-template strand is missing or mismatched. Under the latter conditions, much of the RNA product remains associated with the template. The latter results suggest that proper resolution of the transcription bubble at its trailing edge and/or displacement of the RNA product are required for termination at this class of signal.
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INTRODUCTION |
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A variety of signals have been found to modulate the process of transcript elongation. In general, these have been categorized as falling into the following three classes: pause sites, which temporarily halt the RNA polymerase (RNAP)1 but subsequently allow resumption of transcription; termination signals, which cause release of the RNA and dissociation of the transcription complex; and arrest sites, at which the RNAP may be halted for a prolonged period but may escape by cleavage and subsequent elongation of the transcript (for review, see Refs. 1 and 2). Among the termination signals, the best characterized involve the formation of a stem-loop structure in the nascent RNA (3-5). Although there have been reports of pause, arrest, or termination signals that do not involve the formation of a structured RNA (see for example Ref. 6), these signals have been less well studied. In this work, we have characterized a sequence-specific pause/termination signal for T7 RNAP and have identified the elements that are required for its function.
Two types of signals are known to cause pausing and/or
termination by T7 RNAP (7, 8). Class I terminators, typified by the
signal that is present in the late region of T7 DNA (T), encode RNAs
that have the potential to form stable stem-loop structures followed by
a run of U residues. These features are reminiscent of many intrinsic
terminators utilized by Escherichia coli RNA polymerase, and a number of bacterial termination signals have been shown to cause T7 RNAP to terminate (8-13). Although the members
of this class encode RNAs that share a typical secondary structure,
they exhibit little sequence homology.
A second type of termination signal recognized by T7 RNAP was first identified in the cloned human prepro-parathyroid hormone (PTH) gene (8, 14). These signals (class II signals) do not encode RNAs with an apparent consistent secondary structure but share a common sequence (ATCTGTT, in the non-template strand (8, 15, 16); this work). Additional members of this class were subsequently identified in the concatemer junction of replicating T7 DNA, in the E. coli rrnB T1 terminator, in a cDNA copy of the intergenic region of vesicular stomatitis RNA, in adenovirus DNA, and possibly in bacteriophage lambda DNA (15-19). Whereas some of these signals function as termination sites, others serve as pause sites that terminate T7 RNAP efficiently only in the presence of T7 lysozyme (an inhibitor of T7 RNAP) or when transcribed by mutant RNAPs that show increased sensitivity to lysozyme (15, 18). Other mutant T7 RNAPs have been identified that fail to recognize class II signals yet continue to recognize class I signals (8, 17), indicating that termination at class I and class II sites involves non-equivalent mechanisms. Recognition of class II signals is important for T7 development, as a mutant polymerase that does not recognize a class II pause site found in the concatamer junction of replicating T7 DNA is unable to support T7 growth, apparently due to a block in the processing and packaging of DNA into phage particles (15, 17, 20).
In this work, we have characterized the prototypical class II termination signal found in the PTH gene with the intention of identifying the elements that are required for its function and illuminating its mode of action. We found that termination at this signal is sequence-specific and requires the presence of a conserved sequence in duplex DNA, 7 bp upstream of the site of termination. Each of the nucleotides within this sequence (ATCTGTT in the non-template strand) is critical for function, as any substitutions abolish termination.
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EXPERIMENTAL PROCEDURES |
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DNA and Enzymes-- All plasmids were constructed by standard procedures (21); sequence files are available on request. DNA oligomers were synthesized by Macromolecular Resources (Colorado State University) and purified by low pressure reverse phase chromatography. Prior to transcription, plasmid templates were digested with HindIII, treated with proteinase K, extracted with phenol and chloroform, and precipitated with ethanol (21). Histidine-tagged versions of wild type T7 RNAP and the mutant RNAPs del172-3 (17) and X19 (20) were purified as described previously (22).
To construct pBH182, a synthetic 52-nt DNA oligomer (TCGAATTCAATTAATACGACTCACTATAGGGAGACCACAACCATGGTACCTG) that contains a consensus T7 promoter (underlined) was annealed to its complement, digested with EcoRI and KpnI, and inserted into the EcoRI and KpnI sites of pUC19. To construct plasmids pBH183, pBH184, pBH194, pBH195, pBH196, pBH2O2, pBH220, and pBH221, synthetic DNA oligomers having the sequences shown in Table I were annealed to a complementary oligomer that resulted in BamHI- and SalI-compatible ends and were inserted into the BamHI and SalI sites of pBH182. Modified PTH signals having individual bp substitutions were constructed by polymerase chain reaction mutagenesis using pBH220 as a template. The "upstream" primer (DL31, GTGAATTCAATTAATACGACTCACTATAG) included part of the T7 promoter (underlined) and an EcoRI site (italics). The "downstream" primers (DL32-DL50, GCTCTAGATATCAAAACAGATGATCCCCGGGTACCA) included the PTH signal (underlined) and an XbaI site (italics). With the exception of DL33 (which contains the wild type sequence) each of these primers introduced a single bp substitution into the PTH signal, as noted in Table II. Polymerase chain reaction was performed using a PrimeZymeTM kit (Biometra) according to the recommendations of the manufacturer. Reactions were preincubated at 95 °C for 4 min and subjected to 30 cycles of 92 °C for 1 min, 42 °C for 1 min, and 72 °C for 30 s. The products were digested with EcoRI and XbaI and cloned into the corresponding sites of pUC19; the DNA sequence of each cloned interval was confirmed using chain terminating ddNTPs. To prepare synthetic templates the combinations of oligomers indicated 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; 1 mM dithiothreitol; 0.05% Tween 20) (23), and the samples were heated to 70 °C for 10 min and then cooled slowly. The sequences of the oligomers were as follows: BH120, ATTCAATTAATACGACTCACTATAGGGAGACCACAACCATGGTGATCTTGCCATCTGTTTTCTTGCAAGATATCGGGCCG; BH122, ATTCAATTAATACGACTCACTATAGGGAGACCACAACCATGGTGATCTTGCGGCAACAACGATTGCAAGATATCGGGCCG; BH123, CGGCCCGATATCTTGCAATCGTTGTTGCCGCAAGATCACCATGGTTGTGGTCTCCCTATAGTGAGTCGTATTAATTGAAT; BH135, CGGCCCGATATCTTGCAAGAAAACAGATGGCAAGATCACCATGGTTGTGGTCTCCCTATAGTGAGTCGTATTAATTGAAT; BH136, ATTCAATTAATACGACTCACTATAGGGAGACCACAACCAACCACTAGAACGCATCTGTTTTCTTGCAAGATATCGGGCCG; BH146, ATTCAATTAATACGACTCACTATA; BH147, ATTCAATTAATACGACTCACTATAGGGAGACCAC; BH148, CTTGCCATCTGTTTTCTTGCAAGATATCGGGCCG; BH149, ATTCAATTAATACGACTCACTATACCCTCTGGTGAACCATGGTGATCTTGCCATCTGTTTTCTTGCAAGATATCGGGCCG; BH150, ATTCAATTAATACGACTCACTATAGGGAGACCACAACCATGGTGATCTTGCCATCTGTTTTC; BH151, ATTCAATTAATACGACTCACTATAGGGAGACCACAACCATGGTGATCTTGCCATCTGTTTTCTTGC; BH152, AACCATGGTGATCTTGCCATCTGTTTTCTTGCAAGATATCGGGCCG; AK1, ACACGACGAACCATGGTGATCTTGCCATCTGTTTTCTTGCAAGATATCGGGCCG; AK2, ATTCAATTAATACGACTCACTATAGGGAGACCACGCTGCAAT; SCB84, CTTGCGGCAACAACGATTGCAAGATATCGGGCCG.Transcription Assays--
Unless otherwise noted, transcription
reactions were 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
(specific activity of 800 Ci/mmol; NEN Life Science Products) or 4 µCi of [
-32P]GTP (specific activity of 6,000 Ci/mmol; NEN Life Science Products); 10-20 ng of RNA polymerase, 1 µg of plasmid template or 50 nM synthetic DNA template,
and 4 units of RNasin (Boehringer Mannheim). Reactions were incubated
at 37 °C for 15 min, and the products were analyzed by
electrophoresis in polyacrylamide gels containing 7 M urea
as described previously (22). 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 termination efficiency was calculated as: termination
efficiency = (termination product)/(termination product + run-off
product) taking into account the base composition of the individual
transcripts.
RNase Mapping--
To prepare RNA size markers, plasmid pBH183
digested with HindIII was transcribed with the mutant T7
RNAP del172-3 in a volume of 40 µl containing 4 µg of
DNA, 100 ng of del172-3 RNAP, 80 µCi of
[-32P]GTP, 0.2 mM GTP, and 0.5 mM ATP, CTP, and UTP under standard conditions (see above)
at 37 °C for 30 min. DNase was then added (1 unit, Promega), and the
sample was incubated for 15 min at 37 °C. To generate specific
fragments, 18 ng of RNase A (Boehringer Mannheim) or 2 units of RNase
T1 (Boehringer Mannheim) were added to 15 µl of the reaction, and the
sample was incubated at room temperature for 1 min. The reactions were
terminated by the addition of stop buffer, and the products were
resolved by electrophoresis in 20% polyacrylamide gels in the presence
of 7 M urea, along with termination and run-off products
made by transcription of pBH183 with wild type T7 RNAP under standard
conditions using [
-32P]ATP as the label, as described
previously (Ref. 22; see Fig. 2).
Single-round Transcription Reactions and Gel Shift
Assays--
All reactions were carried out in 40 mM
K-HEPES, pH 7.8; 6 mM dithiothreitol; 10 mM
MgCl2. The template strand oligonucleotide BH123 (500 nM) was end-labeled with T4 polynucleotide kinase and [-2P]ATP (21), and the kinase was inactivated by
heating to 80 °C. To construct double-stranded templates, the
labeled template strand oligomer was mixed with the indicated
non-template strand oligomer (see Fig. 7) at a concentration of 250 nM (each oligomer), and the reactions were heated to
95 °C and cooled slowly to room temperature. The annealed template
(1 µl) was mixed with 2 µl containing 125 nM T7 RNAP, 1 mM GTP, and 1 mM ATP (in the same buffer), and
the samples were incubated at 30 °C for 2 min. The reactions were then completed by the addition of 2 µl containing 2.5 mg/ml sodium heparin (Life Technologies, Inc.), and 1 mM CTP and UTP.
After further incubation for 2 min the reactions were terminated by the
addition of 5 µl of stop buffer containing 0.2% SDS, and the products were analyzed by gel electrophoresis under non-denaturing conditions (24). The gels (1.5 mm × 14 cm × 14 cm, 10%
acrylamide:bisacrylamide (19:1), 1× TBE, 4 mM
Mg(OAc)2, 0.1% SDS, 0.1% ammonium persulfate, and 0.01%
TEMED) were pre-equilibrated at 60 V for 1 h at room temperature.
Following loading of the samples, electrophoresis was continued at 60 V
for 12 h. Samples to be analyzed under denaturing conditions were
mixed with an equal volume of sample buffer (6 M urea; 10 mM Na3EDTA, pH 8.0; 0.01% xylene cyanol; and
0.01% bromphenol blue) and analyzed as described previously (22). Products were visualized by exposing the wet gels to film at
70 °C or to a PhosphorImagerTM screen, as described above.
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RESULTS |
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Mapping of the PTH Terminator--
The PTH termination signal had
previously been localized to a 31-bp segment that extends 24 bp
upstream and 7 bp downstream from the termination site (8). To define
the sequences that are essential for PTH function, a series of
synthetic oligomers that include portions of this interval were cloned
downstream from a T7 promoter (Table I).
Transcription of these plasmid templates in vitro (Fig.
1) revealed that replacement of PTH
sequences with plasmid sequences as close as 16 bp upstream from the
termination site still allowed full signal function (i.e.
pBH196). However, the replacement of one additional residue (C to G at
15 in pBH184) abolished terminator function. Thus, the minimal
upstream boundary of the PTH terminator is 15 bp from the termination
site.
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Effects of Base Pair Substitutions in the PTH Signal-- The analysis summarized in Table I identified a 10-bp interval (CATCTGTTTT in the non-template strand) that functions to provide efficient termination. A comparison with other class II signals reveals a 7-bp sequence (underlined) found in all of these signals (Table I) (15). To determine the importance of individual base pairs to PTH function, we constructed modified signals having single base pair substitutions and examined their ability to terminate T7 RNAP (Fig. 3 and Table II). In this analysis, we did not investigate the effects of substitutions of the four U residues that lie downstream from the conserved sequence, as the importance of these residues to terminator function had already been demonstrated (Refs. 8 and 14 and see above). Strikingly, any substitutions of base pairs within the conserved sequence resulted in strong inhibition of signal function, indicating a strict requirement for sequence conservation in this region. Conservation of the bp just upstream of the underlined sequence (CATCTGTT), although important, is not absolutely required, as all substitutions except G at this position were well tolerated. We therefore conclude that the minimal sequence required to cause efficient termination by the PTH signal is HATCTGTTTT (where H is A, C, or T).
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Both Strands of the DNA Duplex Are Required for Termination at the
PTH Signal--
Whereas transcription by T7 RNAP requires a promoter
that is double-stranded in the binding region, the presence of the
non-template (NT) strand is not required in the initiation region of
the promoter, or downstream, for RNA synthesis to proceed (26, 27).
This feature of T7 transcription allows the importance of the NT strand to termination to be assessed. We and others (8, 16) had previously
reported that the class I termination signal, T, continues to
function when present in a single-stranded template. In contrast, as
shown in Fig. 4 and previously reported
by Hartvig and Christiansen (16), the class II PTH signal is not
utilized efficiently when it is present only in the template (T)
strand.
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Gaps or Heteroduplex Regions Upstream from the PTH Signal Prevent Termination-- A number of observations suggested to us that proper displacement of the RNA product from the template might be important for PTH terminator function. First, as noted above, T7 RNAP fails to terminate at the PTH signal when transcribing a single-stranded DNA template. Second, Ikeda and Richardson (28) had previously reported that a proteolytically modified form of T7 RNAP which fails to utilize the PTH terminator (8) may be defective in displacing the RNA product. Finally, Mead2 has found that utilization of the PTH terminator is decreased about 10-fold on a negatively supercoiled template as opposed to a linear template, suggesting that the stability of the double-stranded template (which may affect product displacement) influences termination efficiency.
Reannealing of the T and NT strands at the trailing edge of the elongation complex is likely to be important in displacing the RNA product or preventing its reannealing to the T strand after displacement. It might be expected, therefore, that lack of a homologous NT strand in a localized region of duplex DNA could allow the RNA product to anneal to the T strand, resulting in the formation of an extended RNA:DNA hybrid that would be propagated as the elongation complex proceeds downstream (see Fig. 7). We therefore examined the effects of gaps in the NT strand or of locally unpaired regions, when these discontinuities in template structure were placed upstream from the PTH terminator. As shown in Fig. 5, the presence of a heteroduplex region (lane 5) or of a gap (lane 8) upstream from the PTH signal greatly diminishes or abolishes recognition of the signal, even though the PTH sequence is present in both strands of the DNA. This is particularly true for termination at the major site of termination (T2). Note that the presence of a "nick" in the NT strand does not prevent termination (lane 9) nor does the presence of unpaired ends of the NT strand on either side of a nick (lanes 10 and 11), indicating that a more extended disruption in the local pairing of the duplex DNA upstream of the PTH signal is required to cause this effect. The absence of the NT strand downstream of the PTH sequence reduces termination at the major site (T2) but allows continued termination at T1 (lanes 6 and 7). In these constructs, the DNA at T1 is double-stranded, whereas at T2 the NT strand is either absent (lane 6) or extends just to the site of termination (lane 7). These results indicate that efficient termination at T2 requires the presence of duplex DNA in this region, as well as in the 10-bp PTH element that lies upstream.RNA Displacement Versus Termination-- As noted above, a possible explanation for the failure of templates having interrupted (gapped or heteroduplex) NT strands to terminate efficiently is that improper displacement of the RNA product interferes with signal function. We have directly explored the potential of such templates to form RNA:DNA hybrids by means of a single round transcription experiment, as shown in Fig. 6. Here, a T strand oligomer (labeled at its 5' end with 32P) was annealed to different NT oligomers, resulting in template constructs having various conformations downstream from a T7 promoter. The labeled templates were incubated with RNAP in the presence of GTP and ATP to allow the formation of an elongation complex, and the remaining substrates (CTP and UTP) were then added along with heparin (which inactivates free RNAP but not RNAP in an elongation complex). Changes in the conformations of the templates before and after transcription were examined by dissociating the RNAP with detergent (SDS) and resolving the templates by electrophoresis under non-denaturing conditions. Transcription of a completely duplex, double-stranded DNA (template 7) did not result in a change in mobility of the template. Transcription of a template that is single-stranded downstream of the start site for transcription (template 2) resulted in a nearly complete shift in mobility of the template from its original position to slower migrating forms, consistent with the formation of an RNA:DNA hybrid. The two distinct slower migrating species formed under these conditions may arise from annealing of short abortive RNAs to the T strand in the initiation region (resulting in the smaller of the two high molecular weight forms) or annealing of full-length RNA to the T strand (resulting in the larger of the high molecular weight forms). Transcription of double-stranded templates having locally unpaired regions (i.e. a gap, template 4, or a "bubble," templates 5 and 6) also resulted in a decrease in the amount of template migrating at the pre-transcription position, as did transcription of a template that is double-stranded to +11. However, in the latter cases only a single discrete band of lower mobility was observed. These findings are consistent with the interpretation that the smaller of the two high molecular weight forms that are observed upon transcription of the single-stranded template (template 2) results from annealing of abortive RNAs, and that the presence of the NT strand in the initiation region in the latter constructs prevents this from occurring.
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DISCUSSION |
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We have characterized the prototypical class II signal found in the cloned human PTH gene (14) and have determined that a 10-bp DNA element (HATCTGTTTT in the nontemplate strand, where H is A, C, or T) is necessary and sufficient to cause efficient termination by T7 RNAP. The preferred site of termination occurs 5 nt downstream from this element, but conservation of the sequence in the region of termination, and in particular the nucleotide at the site of termination, is not required for signal function. Although the terminator functions in a context-independent manner, it is orientation-dependent and is utilized only when transcribed in the direction indicated (8).
Because class II termination signals were first identified in a cloned human gene, their significance in phage replication was unclear. However, subsequent studies have revealed the presence of a class II signal in the concatamer junction of replicating T7 DNA and of related phages such as T3, SP6, and K11 (15, 20). Recognition of this signal is required for T7 growth, as polymerase mutants that do not utilize the signal are unable to support phage development, apparently due to failure to process and package the newly replicated DNA into phage particles (15, 17, 20).
All class II signals are related to a 7-bp conserved sequence (underlined above) that is contained within the 10-bp PTH element (Refs. 16 and 17, and see Table I). However, the efficiency of termination among members of this class is variable. Some signals (such as the PTH signal) function as intrinsic terminators at which RNA release is rapid and efficient, whereas others (such as the sequence found in the concatamer junction of replicating T7 DNA) function primarily as pause sites at which termination is enhanced in the presence of T7 lysozyme or when transcribed by LH mutant T7 RNAPs (15, 17, 18). The efficiency of termination at class II signals appears to depend upon sequences that flank the conserved sequence, and in particular the U run that overlaps the 3' end of the sequence and extends downstream. Substitution of these four U residues in the PTH signal with GCGC had previously been shown to prevent termination (14), and in this work we have found that shortening the U run weakens or abolishes termination. Elsewhere, we have reported that a PTH signal in which the U run has been shortened (i.e. pBH221) functions as a pause site, much like the signal in the concatamer junction of replicating T7 DNA (15).
Unlike class I signals, class II signals do not encode an RNA with an
apparent secondary structure immediately upstream from the termination
site. This observation suggests that although the structure of the
nascent RNA is critical for class I signal function, it is not
important for termination at class II signals. This conclusion is
supported by the observation that substitution of rGMP with rIMP in the
transcript (which destabilizes secondary structure in the RNA)
abolishes termination at the class I signal, T, but does not affect
termination at class II signals (16).
Some elements of the duplex DNA near or around the termination signal appear to be essential for class II terminator function. Thus, unlike class I terminators, class II terminators do not function efficiently when present in a single-stranded template or when present in only the template or non-template strand (i.e. in a local heteroduplex region; Ref. 16 and this work). Further evidence that the stability of the DNA duplex in the PTH signal is important comes from observations that replacement of dGMP residues with dIMP residues in the NT strand abolishes termination (16) and from the decreased termination efficiency observed on negatively supercoiled templates.2
Significantly, the presence of a gap or a heteroduplex region upstream from the PTH signal prevents termination. We interpret this effect to suggest that failure of the NT strand of the DNA to reanneal to the T strand at the trailing edge of the transcription bubble allows the formation of a more extended RNA:DNA hybrid and that this effect is propagated downstream as elongation proceeds. Because duplex DNA is required for PTH signal function, continued failure to resolve the transcription bubble under these circumstances prevents termination (see Fig. 7).
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We have shown that a gap or a heteroduplex region of 10-12 nt is sufficient to cause this effect and that such discontinuities may be located as far as 18 nt upstream from the PTH signal and still prevent termination. Further experiments will be required to determine the minimal length of the discontinuity that causes this effect and the distance over which the effect may be propagated. In earlier work, Daube and von Hippel (24) explored the ability of T7 RNAP to extend an RNA primer hybridized to the template strand of a heteroduplex bubble and found that T7 RNAP actively displaces the primer and the RNA product, releasing the extended product at the end of the transcription cycle. In this work, we utilized templates that are either completely single-stranded downstream from a promoter region or in which there are substantial gaps or heteroduplex regions. We favor the view that T7 RNA polymerase can actively displace the nascent RNA product when transcribing duplex DNA (in agreement with Daube and von Hippel) but that lack of a complementary NT strand may lead to incomplete displacement of the product or allow it to reanneal to the T strand at the trailing edge of the elongation complex (see Fig. 7).
A number of mechanisms could account for the requirement that the PTH signal must be present in both strands of the DNA. For example, recognition of the signal might occur only in double-stranded DNA, either due to base-specific recognition of the sequence in a helical context or in response to a special conformation of the DNA (which of course is also sequence-dependent). In this regard, it is interesting to note that the disposition of the conserved class II sequence relative to the site of termination (spanning an interval 6-15 nt upstream from the termination site) is the same as the disposition of the binding domain of the T7 promoter relative to the start site for transcription (29, 30). The availability of modified PTH signals that fail to cause termination may allow the selection of mutant T7 RNAPs that can utilize these signals, thereby helping to map the region of the RNAP that is responsible for signal recognition.
Alternatively, the NT strand might not play a direct role in signal recognition but might be required merely to ensure proper displacement of the RNA product. Such a situation could arise either because signal recognition involves interactions between the RNAP and the nascent RNA or because collapse of the transcription bubble is required to complete the termination event. In support of the latter hypothesis, we have found that the presence of the PTH signal in the T strand alone results in a significant decrease in the production of run-off products without giving rise to efficient termination, suggesting that under these conditions the signal acts as a pause site. However, two observations argue against a wholly passive role for the NT strand in termination. First, replacement of rGMP with rIMP in the transcript (which should decrease the stability of the RNA:DNA hybrid and enhance product displacement) does not restore termination on a single-stranded template (16). Additionally, we note that after the first round of transcription on a single-stranded DNA template, newly synthesized RNA would remain hybridized to the T strand. If the role of the NT strand were merely to displace the product, then the RNA formed in the first (and subsequent) cycle(s) should be able to fulfill this function. The observation that termination does not occur during multiple rounds of transcription on single-stranded templates argues against a passive role for the NT strand. (However, because an NT strand that is in the form of RNA might not interact with the RNAP in the normal fashion, we cannot exclude this possibility.)
Little is known about the properties of the transcription bubble in an elongating T7 RNAP complex. We had previously observed that when T7 RNAP "slides" through a poly(dA) tract in the template strand, the minimal number of UMP residues incorporated is 8-12 nt, indicating that the length of the RNA:DNA hybrid under these conditions may be 8-12 bp (7). Consistent with this, Tyagarajan et al. (31) reported that the newly synthesized RNA product must extend 10 nt from the site of polymerization before it becomes accessible to a self-cleaving hammerhead structure encoded in the same transcript, suggesting that this length of RNA is sequestered within an RNA:DNA hybrid or is otherwise constrained within the elongation complex.
By using a topoisomerase relaxation assay to measure DNA unwinding during transcription, it has recently been reported that the extent of unwinding in a halted T7 elongation complex is 10-14 bp.3 However, the length of the bubble is dynamically determined by competition between the rate at which it forms (at the leading edge) and the rate at which it collapses (at the trailing edge). The former parameter depends upon the rate of elongation, and it has been found that during rapid elongation the length of the bubble may be up to 50% greater than in a halted complex.3 In a dynamic model, the topology of the template and the transcription complex are also expected to affect the size of the bubble. Thus, the length of the RNA:DNA hybrid would be longer on a supercoiled template where RNA displacement is less favored and would also be affected by the negative supercoiling that accumulates behind a rapidly transcribing complex due to inhibition of free rotation by viscous drag of a lengthy RNA product (33).3 The latter effect may explain the observation that termination at the minor site in the PTH signal (T1) is enhanced when transcribing a short synthetic DNA template as opposed to transcription from a larger, plasmid template (compare Fig. 4 with Figs. 1 and 3).
The proteolytically nicked form of T7 RNAP and mutant enzymes that are altered in the proteolytically sensitive region (all of which fail to terminate at class II signals) exhibit a variety of phenotypes that may be related to the maintenance or resolution of a transcription bubble. These include a reduced ability to maintain an open complex during initiation, a reduced ability to bind RNA, a decreased stability in a halted elongation complex, and a decrease in processivity and RNA displacement (especially on supercoiled templates) (17, 28, 34, 35).3,4 As proper resolution of the transcription bubble appears to be essential for termination at class II signals, alterations in the manner in which these enzymes resolve the bubble are likely to account for their failure to utilize this class of signal.
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ACKNOWLEDGEMENTS |
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We are grateful to Ray Castagna for technical assistance and Rita Gould and Roseann Lingeza for secretarial assistance. We thank F. W Studier, Asis Das, Sergei Borukhov, David Mead, Rui Sousa, and Konstantin Severinov for valuable discussions.
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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.
§ These authors contributed equally to this work.
¶ Present address: Howard Hughes Medical Institute, Dept. of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208.
Present address: Dept. of Hematology and Oncology, Wexner
Pediatric Research Institute, Children's Hospital, 700 Children's Drive, 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., P. O. Box 44, Brooklyn, NY 11203-2098. Tel.: 718-270-1238; Fax: 718-270-2656.
1 The abbreviations used are: RNAP, RNA polymerase; bp, base pair(s); nt, nucleotide(s); PTH, prepro-parathyroid hormone; LH, lysozyme-hypersensitive; TEMED, N,N,N',N'-tetramethylethylenediamine; NT, non-template; T, template.
2 D. Mead, personal communication.
3 V. Gopal, W. T. McAllister, and R. Sousa, submitted for publication.
4 P. E. Karasavas and W. T. McAllister, unpublished observations.
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REFERENCES |
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