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* Morse Institute of Molecular Genetics, Department of Microbiology
and Immunology, State University of New York Health Science Center at
Brooklyn, 450 Clarkson Avenue, Box 44, Brooklyn, NY 11203-2098;
and Communicated by F. William Studier, Brookhaven National
Laboratory, Upton, NY, October 5, 2000 (received for review May 25, 2000)
During the early stages of transcription, T7 RNA polymerase forms
an unstable initiation complex that synthesizes and releases transcripts 2-8 nt in length before disengaging from the promoter and
isomerizing to a stable elongation complex. In this study, we used
RNA·protein and RNA·DNA crosslinking methods to probe the
location of newly synthesized RNA in halted elongation complexes. The
results indicate that the RNA in an elongation complex remains in an
RNA·DNA hybrid for about 8 nt from the site of nucleotide addition
and emerges to the surface of the enzyme about 12 nt from the addition
site. Strikingly, as the transcript leaves its hybrid with the
template, the crosslinks it forms with the RNA polymerase involve a
portion of a hairpin loop (the specificity loop) that makes specific
contacts with the binding region of the promoter during initiation.
This observation suggests that the specificity loop may have a dual
role in transcription, binding first to the promoter and subsequently
interacting with the RNA product. It seems likely that association of
the nascent RNA with the specificity loop facilitates disengagement
from the promoter and is an important part of the process that leads to
a stable elongation complex.
Despite a lack of obvious
sequence or structural homology between the single subunit T7 RNA
polymerase (RNAP) and the multisubunit RNAPs, the basic features of the
transcription process are highly conserved among the two groups of
enzyme (reviewed in ref. 1). As is the situation with other RNAPs, T7
RNAP forms an unstable initiation complex (IC) that synthesizes and
releases transcripts 2-8 nt in length (abortive initiation products)
before disengaging from the promoter and isomerizing to a stable
elongation complex (EC). Whereas considerable biochemical and
structural data are available concerning T7 RNAP initiation complexes,
little is known about the transition to an EC or about the properties
of the stable complex. Recognition of the promoter involves a
specificity loop in the RNAP (amino acid residues 739-770) that
projects into the DNA binding cleft and interacts with the binding
region of the promoter, which lies 7 to 11 bp upstream from the active
site (i.e., positions The transformation to a stable EC commences when the nascent RNA has
achieved a length of RNA Polymerase and Templates.
Mutant RNAPs were constructed and purified as described (10, 11). All
RNAPs described here have an N-terminal His6
leader and exhibited normal activity. Templates that allow the
incorporation of UTP analogs at defined positions in the transcript and
the use of immobilized RNAPs to extend the transcript by successive cycles of limited elongation have been previously described (8, 11,
12).
RNA·DNA and RNA·Protein Crosslinking.
To prepare halted elongation complexes, 20 pmol of T7 RNAP was
incubated with an equimolar concentration of template, 0.3 mM GTP, 0.1 mM ATP, and 50 µM UTP analog (see below) in 20 µl of transcription
buffer (40 mM Tris-acetate, pH 7.9/8 mM magnesium acetate/5 mM
Biochemistry
The specificity loop of T7 RNA polymerase interacts first with
the promoter and then with the elongating transcript, suggesting
a mechanism for promoter clearance
,
, Public Health Research Institute, New York, NY 10016
Abstract
Top
Abstract
Introduction
Materials and Methods
RESULTS
Discussion
References
Introduction
Top
Abstract
Introduction
Materials and Methods
RESULTS
Discussion
References
7 to 11) (2, 3). The transition from duplex
DNA in the binding region to open or melted DNA in the initiation
region commences between 5 and 4, and involves an intercalating 
hairpin loop; the template strand is then led down into the active site
by additional contacts with the surface of the enzyme (3). During
abortive initiation, the contacts with the binding region of the
promoter are maintained while the leading edge of the initiation
complex moves downstream, resulting in a more extended footprint of the
complex on the template (4, 5). Packing of the DNA into the complex is
accomplished by "scrunching" of the intervening portion of the
template strand into a hydrophobic binding pocket (6).
9 nt and is accompanied by release of the
upstream promoter contacts (4, 7). However, a transcription complex
with all of the properties of a fully processive EC does not appear to
be formed until after 12-14 nt of RNA have been synthesized (8). It
has been proposed that the progression to a stable EC is triggered by
filling of the template strand binding pocket (6) and/or by
association of the transcript with an RNA product binding site in the
N-terminal domain of the RNAP (9). The work shown here indicates that
interactions between the nascent RNA and the specificity loop are an
important element in this transition.
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
RESULTS
Discussion
References
-mercaptoethanol/0.1 mM EDTA) for 5 min at 37°C. The startup
complexes were immobilized on Ni2+-agarose beads
(Qiagen, Chatsworth, CA) and extended in the presence of limiting
mixtures of nucleoside triphosphates at a final concentration of 10 µM each (11). Samples were chilled on ice, and crosslinking was
activated as described below.
was carried out as described in
(13). This analog has two reactive groups that are activated in the
presence of NaBH4, an aldehyde that may form a
Schiff's base with primary amines in the protein, and an aromatic
bis(2-iodoethyl)amino group linked to the fifth position of pyrimidine,
which forms specific crosslinks to the base to which the analog is
paired in an RNA·DNA hybrid (13).
Peptide Mapping. Hydroxylamine (HA) cleavage. A 2-µl aliquot of crosslinked RNAP was mixed with 30 µl of HA (Sigma) in 6 M urea, 4.5 M lithium hydroxide (pH 10), and incubated at 45°C for 2-4 h. The sample was precipitated with 10% trichloroacetic acid (TCA) at 0°C, washed in 5% TCA, taken up in 10 µl of loading buffer, and resolved by PAGE in 10% gels.
Cleavage with 2-nitro-5-thiocyano-benzoic acid (NTCB) and CNBr. Samples prepared as described above were digested with NTCB and CNBr as in ref. 15. Products of cleavage were resolved either by PAGE in 10% gels or in a 4-12% NuPage Bis-Tris gradient gel using an MES buffer system and Seeblue size markers (Invitrogen), as noted in the figure legends. N-chlorosuccinimide (NCS) cleavage. A 2-µl aliquot of crosslinked material was taken up in 3 µl of 150 mM HCl and mixed with 5 µl of NCS (Sigma; 10 mg/ml in water). After 15 min incubation at room temperature, a fresh portion (5 µl) of NCS was added, and the samples were incubated for an additional 5 min. The reaction was stopped by addition of 5 µl of loading buffer, and the samples were analyzed by electrophoresis as described above.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
RESULTS
Discussion
References
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Characterization of the Elongation Complex. To determine the disposition of the RNA in T7 RNAP elongation complexes, we incorporated into the RNA analogs of UTP that may be crosslinked either to DNA or to protein, and subsequently identified the locations of the crosslinks. To place the analogs at defined positions in the transcript, we took advantage of a modified form of T7 RNAP having a His6 leader at the N terminus (10). Use of the modified RNAP allowed us to immobilize transcription complexes on Ni+2 agarose beads and to "walk" the complexes along the template by successive cycles of washing and incubation with limited mixtures of substrates (Fig. 1). The halted complexes studied here have all made transcripts of at least 15 nt and appear to be true elongation complexes; they are highly stable (half-life over 1 h) and are nearly quantitatively extended during each subsequent cycle, indicating either that they represent true intermediates in the reaction pathway or are readily able to reenter the pathway.
|
) that has been shown
to form crosslinks exclusively with the adenine to which it is paired
in an RNA·DNA hybrid (13). In addition to its ability to form
specific crosslinks with DNA, this analog is also able to form
crosslinks with the protein via a reactive aldehyde group (13). Whereas
crosslinking to the RNAP was observed when the analog was placed at any
position between 1 and 17, efficient crosslinking to the DNA was
observed only when the analog was positioned 1 to 8 nt upstream from
the 3' end of the transcript (i.e., positions 1 to 8). (In this
work, we identify positions in the transcript relative to the
elongating 3' end of the RNA at 1; see ref. 15.) These results
demonstrate that the RNA in an EC remains in close proximity to the DNA
(presumably in an RNA·DNA hybrid) from 1 to 8.
To determine at which point the RNA becomes accessible to the solvent,
we labeled the 3' end of a 17-nt transcript in a stable EC and then
treated the complexes with RNase T1 (Fig.
2). Twelve nucleotides of nascent RNA
were protected in the intact EC, indicating that the transcript does
not emerge from the interior of the RNAP until this point. A similar
conclusion was reached in previous studies by using templates that
direct the synthesis of a self-cleaving hammerhead structure in the RNA
(16). Here, it was observed that 13 nucleotides past the cleavage point
must be synthesized before the transcript can fold and self-cleave,
suggesting that the RNA is not free of steric constraints until this
point.
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1 to 17 formed crosslinks with
the RNAP. However, the most efficient crosslinking was observed when
the analog was positioned at 1 and 9. It had previously been shown
that T7 RNAP can bind single-stranded RNA (ssRNA) in a nonspecific
fashion, and that exogenous oligomers of ssRNA or ssDNA are effective
competitors for this binding (9). Based on the RNase T1 protection
experiments described above, it is likely that crosslinking of
transcripts beyond 12 involves a surface binding site(s). Consistent
with this notion, we have found that ssDNA inhibits crosslinking of
transcripts having sUTP positioned at 12 and beyond (not shown).
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-32P]NTPs (Fig. 3). Whereas
transcripts crosslinked from 1 to 7 were extended only poorly,
transcripts crosslinked at 9 and 11 were readily extended (Fig. 3).
The former observation is consistent with the notion that the RNA
nucleotides from 1 to 7 are close to the active site or are
involved in an RNA·DNA hybrid, and that crosslinking would alter
the structure and/or movement of the hybrid. Transcripts crosslinked
at 9 and 11 could be extended by at least 10 nt (Fig. 3),
suggesting either that the element in the RNAP to which the transcript
is crosslinked is flexible, and/or that the RNA is extruded between
the site of its displacement from the template and the site on the
protein to which it is crosslinked. Interestingly, transcripts
crosslinked from 13 to 17 were extended only poorly, perhaps
because the region of the RNAP affected by these crosslinks is crucial
for processive elongation (see Discussion).
Mapping of the Crosslink at 9.
For a number or reasons, the crosslink at 9 was of particular
interest. First, as noted above, it seems likely that the base at this
position is near the point at which the transcript is displaced from
the template. More importantly, it corresponds to the length of the
nascent transcript that is present when the transition from an unstable
IC to a stable EC commences (4, 7, 8). To map this contact in the EC,
we used a combination of conventional protein mapping methods together
with site-directed mutagenesis.
30-kDa
fragments from both the N- and C-terminal regions as well as two
partial digestion products of 60 kDa. As shown in Fig.
4, peptide fragments of these sizes were
labeled by a transcript crosslinked at 9. To discriminate whether it
was the C-terminal or the N-terminal fragment that was being
illuminated, we constructed a mutant T7 RNAP in which the N-terminal
cleavage site was eliminated by substitution of Asn-289 with Asp
(N289D). Digestion of the crosslinked mutant protein resulted in a
labeled 30-kDa fragment but no label in the 60-kDa fragment (Fig. 4). Because the only cleavage site in the mutant protein is the NG at 589, the crosslink with RNA at 9 must involve amino acid residues in the
30-kDa fragment between 589 and 883.
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9 crosslink further, the 13-kDa and 18-kDa NTCB
fragments from the wild-type (WT) enzyme were purified by gel
electrophoresis and subsequently cleaved with NCS or CNBr. As shown in
Fig. 5, each treatment produced a mixture
of labeled products resulting from partial digestion. The only
consistent interpretation of these data is that the smallest labeled
band apparent after NCS digestion represents the interval W737-W797, and the smallest labeled band after CNBr treatment represents the
interval from A724 to M750. (In these experiments, the labeled bands
migrate somewhat more slowly than protein markers of equivalent size
because of the crosslinked RNA.) Thus, the 9 crosslink must lie
between residues 737 and 750. This assignment was corroborated by
cleavage of the 30-kDa HA fragment with NTCB, CNBr, and NCS (not
shown).
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Discussion |
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Abstract
Introduction
Materials and Methods
RESULTS
Discussion
References
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The specificity loop of T7 RNAP (which comprises amino acid
residues 739-770) projects into the DNA binding cleft of the RNAP and
makes specific contacts with the upstream region of the promoter during
binding and initiation. The 7-aa interval between Q744 and M750 to
which the crosslink at 9 of the RNA in an EC is made corresponds to
one arm of this loop and encompasses residues that are directly
involved in promoter recognition. These residues include N748, which
interacts with the base pairs at 10 and 11 of the promoter DNA, and
R746, which interacts with the base pair at 7 (2, 3). The observation
that the transition from IC to EC commences when the RNA achieves a
length of 9 (4, 7, 8), together with the observation that the
nucleotide at 9 in the RNA in an EC forms crosslinks with the
promoter-recognition region of the polymerase, suggests that an
interaction between the nascent transcript and the specificity loop
plays an important role in promoter clearance and/or stabilizing the
EC. Perhaps the growing RNA chain disrupts the interaction between the
specificity loop and the promoter, or stabilizes a conformation of the
loop that inhibits its re-association with the DNA.
Four separate crystal structures have been solved for T7 RNAP: the apoenzyme, the enzyme complexed with T7 lysozyme (an inhibitor of T7 RNAP), a binary complex of the RNAP bound to its promoter, and an initiation complex in which the first three nucleotides of RNA have been synthesized (3, 6, 18, 19). As yet, there is no information with regard to the structure of an elongation complex such as we have characterized here.
In earlier studies, Jeruzalmi and Steitz (19) modeled a putative RNA·DNA hybrid into the structure of a T7 RNAP·lysozyme complex by homology with the TaqI DNA polymerase primer/template complex, and found that the binding cleft could neatly accommodate 6-8 bp of hybrid with little steric clash. However, in a more recently solved structure of an initiation complex formed in the presence of GTP (which allows incorporation only of the first three G residues), Cheetham and Steitz (6) observed a different trajectory for the 3-bp RNA·DNA hybrid than predicted in the earlier study, and noted that further extension of the hybrid would result in a steric clash with the N-terminal domain. Furthermore, they noted that the base at the 5' end of the transcript appeared to be "peeling off" from the template (i.e., was not involved in true Watson-Crick base pairing). Based on these observations, the authors suggested that the RNA·DNA hybrid could not exceed 3 bp in the IC. The exit pathway for the displaced RNA proposed in the latter study is not consistent with the results obtained here (Fig. 6), but may correspond to the path for poly(G) products that are synthesized by transcript slippage at promoters that initiate with +1 GGG (7) or for short products that are released during abortive initiation.
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In considering the possible position of the RNA·DNA hybrid in an
elongation complex, we repeated the earlier strategy of Jeruzalmi and
Steitz (19) by homology modeling the TaqI DNA polymerase primer/template complex (20) into the T7 RNAP initiation complex, superimposing the highly conserved residues D537, D812, and Y639 in the
active site of T7 RNAP with the corresponding residues in
TaqI DNAP (Fig. 6). As in the earlier studies (19), we
observed few steric clashes between the RNA·DNA hybrid and
residues in the binding cleft. Although the position (and shape) of the
hybrid should be considered tentative, the model predicts that the
transcript nucleotide at 9 would be near the region in the
specificity loop to which it forms a crosslink. Furthermore, the
trajectory of the displaced RNA would direct it toward a previously
identified surface binding site in the N-terminal domain (21).
As noted above, the transition to a stable EC commences when the RNA
has achieved a length of 9 nt, but the transition is not completed
until after 12-14 nt have been synthesized (8). The later stages in
this process may involve binding of the emerging transcript to the
surface binding site. Preliminary experiments have shown that
transcripts crosslinked at 14 are attached to a region of the RNAP
that lies near the HA cleavage site at position 289, which is
consistent with this expectation (unpublished observations). A number
of mutations that affect processivity and termination map to this
region of the RNAP (22-24), which may explain why crosslinking of
transcripts beyond 13 prevents further elongation.
The model shown in Fig. 6 suggests the possibility that the specificity loop may continue to be involved in displacement of the transcript and resolution of the transcription bubble after the polymerase has cleared the promoter, and might also monitor the DNA and/or RNA for sequences that are involved in termination or pausing (25). Clearly, substantial rearrangements would need to occur during the transition from an IC to an EC in order to accommodate the model proposed. The notion that significant structural alterations occur during isomerization is consistent with a variety of experimental data in the T7 system, as well as results with multisubunit RNAPs (1, 15, 26-29). In further support of the proposed position of the RNA·DNA hybrid in the T7 EC, we note that many of the structures that are involved in interactions with the primer/template in the pol I family of DNAPs are conserved in T7 RNAP (19), suggesting that these regions may be involved in similar functions in both RNA and DNA polymerization.
The overall organization of the T7 elongation complex described here bears a remarkable similarity to the organization of ternary complexes formed by the multisubunit RNAPs (15). Thus, for both types of RNAP, the RNA·DNA hybrid is proposed to be 8-9 bp in length, and the RNA does not emerge to the surface of the enzyme until 12-14 nt have been synthesized. In this work, we have proposed a role for the specificity loop of T7 RNAP in binding the nascent transcript and resolving the trailing edge of the transcription bubble. A similar role has been proposed for the "rudder" element in Escherichia coli RNAP (15, 30). (We prefer the more nautically correct term of marlinespike, a tool that is used to separate the strands of a rope during splicing.) Interestingly, we note the presence of highly conserved basic residues (arginine and lysine) on the surface of the rudder that faces the putative RNA·DNA hybrid in E. coli RNAP, and the presence of a conserved arginine residue (R746) in the region of the specificity loop of T7 RNAP that interacts with the nascent transcript. Whereas the rudder is not directly involved in promoter interactions, a coiled-coil structure that projects from this element, and the flexible "flap" under which the nascent RNA emerges after its displacement, are both thought to interact with the sigma subunit (the transcription specificity factor that is involved in promoter recognition) (15, 29). Thus, as in the case of T7 RNAP, interactions of the nascent RNA with the rudder or the flap may trigger release of promoter interactions and/or stabilize the EC.
The observation that transcripts crosslinked at 9 may be further
extended, perhaps by looping out of the RNA, suggests that the
organization of the EC may be somewhat flexible. This finding has
potential implications for models of pausing and termination, as
conditions that slow the transit of the emerging transcript over the
surface of the RNAP while maintaining a high rate of polymerization
might also lead to extrusion of the RNA, providing an opportunity for
the transcript to contact additional surfaces of the enzyme.
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Acknowledgements |
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We thank Ray Castagna, Manli Jiang, and Dr. Michael Anikin for technical advice and assistance, and Dr. Vadim Nikoforov for critical comments on the manuscript. This work was supported by National Institutes of Health Grants GM381477 to W.T.M., GM54098 to S.B., and GM30717 and GM49242 to A.M.
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Abbreviations |
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RNAP, RNA polymerase; IC, initiation complex; EC, elongation complex; HA, hydroxylamine; NTCB, 2-nitro-5-thiocyanobenzoic acid; WT, wild type; NCS, N-chlorosuccinimide.
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Footnotes |
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Present address: Department of Microbiology and
Molecular Genetics, Life Sciences, Room 166, SUNY Stony
Brook, Stony Brook, NY, 11794.
§ To whom reprint requests should be addressed. E-mail: pogo51{at}aol.com.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.250473197.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.250473197
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References |
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Abstract
Introduction
Materials and Methods
RESULTS
Discussion
References
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G. M. Skinner, C. G. Baumann, D. M. Quinn, J. E. Molloy, and J. G. Hoggett Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase: A SINGLE-MOLECULE VIEW OF THE TRANSCRIPTION CYCLE J. Biol. Chem., January 30, 2004; 279(5): 3239 - 3244. [Abstract] [Full Text] [PDF]
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M. Matsunaga and J. A. Jaehning A Mutation in the Yeast Mitochondrial Core RNA Polymerase, Rpo41, Confers Defects in Both Specificity Factor Interaction and Promoter Utilization J. Biol. Chem., January 16, 2004; 279(3): 2012 - 2019. [Abstract] [Full Text] [PDF]
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B. Gu, V. K. Johnston, L. L. Gutshall, T. T. Nguyen, R. R. Gontarek, M. G. Darcy, R. Tedesco, D. Dhanak, K. J. Duffy, C. C. Kao, et al. Arresting Initiation of Hepatitis C Virus RNA Synthesis Using Heterocyclic Derivatives J. Biol. Chem., May 2, 2003; 278(19): 16602 - 16607. [Abstract] [Full Text] [PDF]
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C. T. Ranjith-Kumar, X. Zhang, and C. C. Kao Enhancer-Like Activity of a Brome Mosaic Virus RNA Promoter J. Virol., February 1, 2003; 77(3): 1830 - 1839. [Abstract] [Full Text] [PDF]
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D. Temiakov, M. Anikin, and W. T. McAllister Characterization of T7 RNA Polymerase Transcription Complexes Assembled on Nucleic Acid Scaffolds J. Biol. Chem., November 27, 2002; 277(49): 47035 - 47043. [Abstract] [Full Text] [PDF]
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 Y. W. Yin and T. A. Steitz Structural Basis for the Transition from Initiation to Elongation Transcription in T7 RNA Polymerase Science, November 15, 2002; 298(5597): 1387 - 1395. [Abstract] [Full Text] [PDF]
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K. Ma, D. Temiakov, M. Jiang, M. Anikin, and W. T. McAllister Major Conformational Changes Occur during the Transition from an Initiation Complex to an Elongation Complex by T7 RNA Polymerase J. Biol. Chem., November 1, 2002; 277(45): 43206 - 43215. [Abstract] [Full Text] [PDF]
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 S. H. Willis, K. M. Kazmierczak, R. H. Carter, and L. B. Rothman-Denes N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases J. Bacteriol., Septe |
(bold). The
complexes were immobilized on Ni2+-agarose beads, and the
transcripts were incrementally extended by sequential cycles of washing
and incubation with the substrates indicated (
