Interrupting the template strand of the T7 promoter facilitates translocation of the DNA during initiation, reducing transcript slippage and the release of abortive products 1 1Edited by M. Gottesman

Jiang, Manli; Rong, Minqing; Martin, Craig T.; McAllister, William T.

doi:10.1006/jmbi.2001.4793

Cited by 36 publications

(42 citation statements)

References 34 publications

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“…A nucleic acid scaffold that allows the formation of an EC by T7 RNAP (TS1͞NT1͞RNA12) has been described (21). Synthetic doublestranded templates that contain a T7 promoter were constructed by annealing complementary T and NT strands together at 70°C for 10 min followed by slow cooling to room temperature (22). The sequences of the NT strands (5Ј-3Ј) for each pair of oligomers were as follows: MJ6͞MJ7 (Fig.…”

Section: Methodsmentioning

confidence: 99%

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages

Temiakov²,

Anikin³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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During the transition from an initiation complex to an elongation complex (EC), the single-subunit bacteriophage T7 RNA polymerase (RNAP) undergoes dramatic conformational changes. To explore the significance of these changes, we constructed mutant RNAPs that are able to form disulfide bonds that limit the mobility of elements that are involved in the transition (or its reversal) and examined the effects of the crosslinks on initiation and termination. A crosslink that is specific to the initiation complex conformation blocks transcription at 5-6 nt, presumably by preventing isomerization to an EC. A crosslink that is specific to the EC conformation has relatively little effect on elongation or on termination at a class I terminator (T ), which involves the formation of a stable stem-loop structure in the RNA. Crosslinked ECs also pause and resume transcription normally at a class II pause site (concatamer junction) but are deficient in termination at a class II terminator (PTH, which is found in human preparathyroid hormone gene), both of which involve a specific recognition sequence. The crosslinked amino acids in the EC lie close to the upstream end of the RNA-DNA hybrid and may prevent a movement of the polymerase that would assist in displacing or releasing RNA from a relatively unstable DNA-RNA hybrid in the paused PTH complex. crosslink ͉ transcriptionA lthough it consists of a single subunit, T7 RNA polymerase (RNAP) carries out all of the steps in the transcription cycle in the same manner as multisubunit RNAPs (1). Therefore, it provides a useful model for studies of the fundamental aspects of transcription. As is the situation with other RNAPs, T7 RNAP forms an unstable initiation complex (IC) that synthesizes and releases short abortive transcripts before it isomerizes to a stable elongation complex (EC) (2). The transition to an EC is accompanied by major structural rearrangements in the protein, resulting in the formation of elements that are important for the stability and processivity of the EC. These include a cavity that can accommodate an RNA-DNA hybrid of 8-9 bp and pores for RNA exit and substrate entry (3, 4). These features of the T7 RNAP EC are similar to those of the multisubunit RNAPs (5, 6).The structural rearrangements leading to an EC largely involve the N-terminal domain of the RNAP (3, 4). In particular, a ''core'' subdomain (residues 72-151 and 206-257) rotates and translocates 35 Å as a rigid body to allow room for the RNA-DNA hybrid. Other regions in the N-terminal domain become refolded to form an element that interacts with the RNA-DNA hybrid and participates in RNA displacement and resolution of the transcription bubble (the ''flap'' subdomain, residues 152-205). A structural element in the C-terminal domain (the specificity loop, residues 739-769) also becomes rearranged during the transition. Whereas in the IC this element is involved in promoter contacts, in the EC it becomes associated with the displaced transcript and forms part of the RNA exit pore (7, 8) (see Fig. 1).T...

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Section: Methodsmentioning

confidence: 99%

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages

Temiakov²,

Anikin³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…A similar increase in 12-and 13-mer transcripts relative to full-length products is observed in transcription from constructs that do not allow normal bubble collapse. An increase in 12-to 13-mer products can be seen in constructs that are nicked on the nontemplate strand in the region of the initially melted bubble, constructs that have an artificially melted (noncomplementary) bubble, and partially single-stranded DNA constructs (15)(16)(17). It has been suggested that improper RNA displacement results in a complex that cannot transcribe well beyond position ϩ13 (16).…”

Section: Are All Complexes Coupled Tomentioning

confidence: 99%

“…An increase in 12-to 13-mer products can be seen in constructs that are nicked on the nontemplate strand in the region of the initially melted bubble, constructs that have an artificially melted (noncomplementary) bubble, and partially single-stranded DNA constructs (15)(16)(17). It has been suggested that improper RNA displacement results in a complex that cannot transcribe well beyond position ϩ13 (16). Artificial bubble scaffolds, such as those that were utilized to trap the elongation complex conformation for crystallographic studies, also lack the ability to properly displace the upstream end of the RNA and are similarly unable to make products longer than a 13-mer with any efficiency (33).…”

Section: Are All Complexes Coupled Tomentioning

confidence: 99%

“…There are fewer abortive products released after translocation to position ϩ10 and lower overall turnover values for complexes artificially stalled beyond position ϩ8 (6). Alternatively, on constructs where promoter release may be inhibited (nicked nontemplate strand or partial single-stranded promoters), an abundance of 12-and 13-mer products relative to runoff (fulllength) products is observed (15)(16)(17). Recent studies suggest that these 12-and 13-mer products may be the result of a failed bubble collapse at the start site.…”

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Cross-linking of Promoter DNA to T7 RNA Polymerase Does Not Prevent Formation of a Stable Elongation Complex

Esposito¹,

Martin²

2004

Journal of Biological Chemistry

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T7 RNA polymerase recognizes a small promoter, binds DNA, and begins the process of transcription by synthesizing short RNA products without releasing promoter contacts. To determine whether the promoter contact must be released to make longer RNA products and at what position the promoter must be released, a mutant RNA polymerase was designed that allows crosslinking to a modified promoter via a covalent disulfide bond. The modifications individually have no measurable effect on transcription. Under oxidizing conditions that produce the protein-DNA cross-link, the complex is able to synthesize short RNA products, strongly supporting a model in which promoter contacts are not lost on translocation through at least position ؉6. However, cross-linked complexes are impaired in promoter escape in that only about one in four can escape to make fulllength RNA. The remainder release 12-and 13-mer RNA transcripts, suggesting an increased energetic barrier in the transition from an initial transcribing complex to a fully competent elongation complex. The results are discussed in the context of a model in which promoter release helps drive initial collapse of the upstream edge of the bubble, which, in turn, drives initial displacement of the 5-end of the RNA.T7 RNA polymerase recognizes a relatively small promoter with near nanomolar affinity (1, 2) and transcribes DNA in a manner that appears to be mechanistically similar to that of the more complex, multi-subunit, eukaryotic and prokaryotic RNA polymerases (3). Because T7 RNA polymerase is a single subunit enzyme capable of carrying out the complete transcription cycle without additional protein cofactors, it is an ideal enzyme to study as a model. Like other DNA-dependent RNA polymerases, T7 RNA polymerase recognizes and binds promoter DNA, melts open an initiation bubble downstream of the promoter, and positions the initial templating bases in the active site to begin the process of transcription (4, 5). After an initial abortive cycling phase characteristic of all RNA polymerases, the enzyme enters a more stable elongation phase after transcription of an ϳ10 -14-mer RNA product (6 -9). Previous studies have suggested that promoter release occurs near the position at which the enzyme switches to the more stable elongation phase, perhaps simultaneously (10). Details concerning the timing and mechanism of promoter release, however, remain unclear.Several studies, including footprinting and fluorescence approaches (2, 10, 11), have shown that polymerase binds the promoter DNA, melts open an initiation bubble positioning the templating (position ϩ1) base in the active site, and then begins transcription, all while maintaining promoter contacts. During the early abortive cycling phase (at least until the polymerase reaches position ϩ6), the promoter contacts remain intact as evidenced by footprinting, although there may be minor perturbations as evidenced by photo cross-linking studies (12). It has also been clearly shown that these promoter contacts are released at some p...

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“…Some time ago, however, it was shown that the immediate connection between the upstream elements and the start site can be replaced by a flexible linkage and initiation is not substantially reduced (the precise start site does, however, become less well-defined and shifts downstream by one base) (6). Subsequently, it was shown that the templating bases can position into the active site in constructs in which the physical connection between the bulk of the upstream single-stranded region and the start site has been lost -the templating strand can be brought to the active site via downstream connections (11). This suggests a model in which the templating bases need only be tethered near the active site.…”

Section: Introductionmentioning

confidence: 99%

Structural Confirmation of a Bent and Open Model for the Initiation Complex of T7 RNA Polymerase

et al. 2007

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T7 RNA polymerase is known to induce bending of its promoter DNA upon binding, as evidenced by gel-shift assays and by recent end-to-end fluorescence energy transfer distance measurements. Crystal structures of promoter-bound and initially transcribing complexes, however, lack downstream DNA, providing no information on the overall path of the DNA through the protein.Crystal structures of the elongation complex do include downstream DNA and provide valuable guidance in the design of models for the complete melted bubble structure at initiation. In the current study, we test a specific structural model for the initiation complex, obtained by alignment of the Cterminal regions of the protein structures from both initiation and elongation and then simple transferal of the downstream DNA from the elongation complex onto the initiation complex. FRET measurement of distances from a point upstream on the promoter DNA to various points along the downstream helix reproduce the expected helical periodicity in the distances and support the model's orientation and phasing of the downstream DNA. The model also makes predictions about the extent of melting downstream of the active site. By monitoring fluorescent base analogs incorporated at various positions in the DNA we have mapped the downstream edge of the bubble, confirming the model. The initially melted bubble, in the absence of substrate, encompasses 7-8 bases and is sufficient to allow synthesis of a 3 base transcript before further melting is required. The results demonstrate that despite massive changes in the N-terminal portion of the protein and in the DNA upstream of the active site, the DNA downstream of the active site is virtually identical in both initiation and elongation complexes.

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Interrupting the template strand of the T7 promoter facilitates translocation of the DNA during initiation, reducing transcript slippage and the release of abortive products 1 1Edited by M. Gottesman

Cited by 36 publications

References 34 publications

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages

Cross-linking of Promoter DNA to T7 RNA Polymerase Does Not Prevent Formation of a Stable Elongation Complex

Structural Confirmation of a Bent and Open Model for the Initiation Complex of T7 RNA Polymerase

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