Structure of a T7 RNA polymerase elongation complex at 2.9 Å resolution

Tahirov, T.H.; Temiakov, Dmitry; Anikin, Michael; Patlan, Vsevolod; McAllister, William T.; Vassylyev, Dmitry G.; Yokoyama, Shigeyuki

doi:10.1038/nature01129

Cited by 226 publications

(328 citation statements)

References 49 publications

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“…The earliest conformational change, observed as a marked decrease in complex stability, occurs at ϩ38. The length of the DNA duplex interacting with the polymerase downstream of the active site was estimated to be 5-8 bp by footprinting (17)(18)(19)(20), consistent with the crystal structure of the T7 elongation complex (21). Thus, in TEC38, more than half of the 8-bp CS would bind to the polymerase as a duplex.…”

Section: Multiple Conformational Changes Along the Class II Terminationmentioning

confidence: 68%

Sequential multiple functions of the conserved sequence in sequence-specific termination by T7 RNA polymerase

Sohn

Kang²

2004

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

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Escherichia coli rrnB terminator t1 contains an RNA hairpin-dependent (class I) and a sequence-specific (class II) termination signal. The latter consists of an 8-bp conserved sequence (CS), TATCTGTT, immediately followed by an 8-bp T rich sequence. In this study, elongation complexes of T7 RNA polymerase at various positions of the class II signal and several mutant signals were obtained by stepwise walking on immobilized DNA templates free of the class I signal. Multiple CS-associated conformational changes were observed, starting at the beginning of the signal and occurring sequentially. When the complexes reach the first base pair of the CS-DNA duplex, which is downstream of the RNA-DNA heteroduplex, their stability, as measured by time-course retention of radiolabeled transcripts, markedly decreases. Further elongation leads to an abrupt change in polymerase-RNA interaction. Crosslinking of the polymerase to a 4-thio-UMP incorporated into RNA 8 nucleotides upstream of the 3 end and just upstream of the heteroduplex is initially strong but diminishes when the polymerase reaches the fourth base pair of the CS. After a further 7-nt elongation, the exposed single-stranded region of nontemplate strand is contracted; RNA in the upstream half of the heteroduplex becomes dissociated, and the CS-DNA duplex is reformed. During the next 5-nt elongation before termination, the CS duplex is prevented from translocation, and the contracted transcription bubble expands only downstream. These findings suggest that the CS duplex plays essential roles by successively binding to polymerase both downstream and upstream of the heteroduplex.photocross-linking ͉ class II termination ͉ stepwise walking of RNA polymerase ͉ RNA-protein interaction ͉ elongation complex stability B acterial RNA polymerases terminate transcription by forming an RNA hairpin followed by a U rich sequence, encoded by a class I termination signal. In contrast, bacteriophage T7 and SP6 RNA polymerases terminate at both class I signals and class II termination signals, the latter consisting of a conserved sequence (CS), HATCTGTT (H designating A, C, or T), followed by a T rich sequence. The class II termination signals found in the Escherichia coli rrnB operon (1), the human preproparathyroid hormone gene (2), and vesicular stomatitis virus DNA (3) terminate phage RNA polymerase transcription, but a CS alone, without a T rich sequence, in the concatamer junction of replicating T7 DNA only causes RNA polymerase to pause (4).The class II sequence-specific termination differs from the class I hairpin-forming termination in several aspects. (i) Class II termination does not depend on formation of an RNA secondary structure; incorporation of IMP in place of GMP into RNA does not affect termination (1, 5). (ii) Class II termination is highly specific in sequence, requiring a perfect CS duplex; introduction of mutations and mismatches abolishes termination (1,5,6). (iii) Introduction of a nick into T7 or SP6 RNA polymerase resulting in N-terminal 20-kDa and C-termi...

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Section: Multiple Conformational Changes Along the Class II Terminationmentioning

confidence: 68%

Sequential multiple functions of the conserved sequence in sequence-specific termination by T7 RNA polymerase

Sohn

Kang²

2004

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

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“…The structures of the initiation and elongation complexes of the single-subunit RNA polymerase from bacteriophage T7 have been determined and show dramatic conformational change in the N-terminal domain of the polymerase Tahirov et al, 2002;Yin and Steitz, 2002). This change disrupts the promoter-binding site in the polymerase, elongates the heteroduplex product binding site from three to eight base pairs, and creates a tunnel through which the emerging RNA transcript can pass in the elongation complex.…”

Section: Common Features Of the Initiation To Elongation Transition Bmentioning

confidence: 99%

“…This change disrupts the promoter-binding site in the polymerase, elongates the heteroduplex product binding site from three to eight base pairs, and creates a tunnel through which the emerging RNA transcript can pass in the elongation complex. Models of the transition between the initiation and elongation phases have implied that the trigger for the conformational change is the force exerted by the elongating heteroduplex product on a helical subdomain of the N-terminal domain of T7 RNA polymerase (Tahirov et al, 2002;Yin and Steitz, 2002;Theis et al, 2004).…”

Section: Common Features Of the Initiation To Elongation Transition Bmentioning

confidence: 99%

The φ29 DNA polymerase:protein-primer structure suggests a model for the initiation to elongation transition

Kamtekar

Berman

Wang

et al. 2006

EMBO J

160

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The absolute requirement for primers in the initiation of DNA synthesis poses a problem for replicating the ends of linear chromosomes. The DNA polymerase of bacteriophage /29 solves this problem by using a serine hydroxyl of terminal protein to prime replication. The 3.0 Å resolution structure shows one domain of terminal protein making no interactions, a second binding the polymerase and a third domain containing the priming serine occupying the same binding cleft in the polymerase as duplex DNA does during elongation. Thus, the progressively elongating DNA duplex product must displace this priming domain. Further, this heterodimer of polymerase and terminal protein cannot accommodate upstream template DNA, thereby explaining its specificity for initiating DNA synthesis only at the ends of the bacteriophage genome. We propose a model for the transition from the initiation to the elongation phases in which the priming domain of terminal protein moves out of the active site as polymerase elongates the primer strand. The model indicates that terminal protein should dissociate from polymerase after the incorporation of approximately six nucleotides.

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“…Effects of mutations in up-and downstream positively charged regions on EC stability. A: Transcripts (13mers) retained ("R") or freed ("F") from halted ECs formed with WT (lanes 1-6), K711C/K713E/K714E (lanes 7-12), K407E/K494E/K332E/K412C (lanes 13-18), or K407E/K412E (lanes 19-24) mutant T7RNAPs in the presence of 0 mM (lanes 1,2,7,8,13,14,19,20), 100 mM (lanes 3,4,9,10,15,16,21,22), or 800 mM (lanes 5,6,11,12,17,18,23,24) NaCl. B: Ratio of Free/Retained transcripts from the experiment in A plotted vs. enzyme and NaCl concentration (error bars give ranges from n=2).…”

Section: Measurement Of Ec Stabilitymentioning

confidence: 99%

Functional Architecture of T7 RNA Polymerase Transcription Complexes

Nayak¹,

Guo²,

Sousa³

2007

Journal of Molecular Biology

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SummaryT7 RNA polymerase is the best-characterized member of a widespread family of single-subunit RNA polymerases. Crystal structures of T7 RNA polymerase initiation and elongation complexes have provided a wealth of detailed information on RNA polymerase interactions with the promoter and transcription bubble, but the absence of DNA downstream of the melted region of the template in the initiation complex structure, and the absence of DNA upstream of the transcription bubble in the elongation complex structure means that our picture of the functional architecture of T7 RNA polymerase transcription complexes remains incomplete. Here we use the site-specifically tethered chemical nucleases and functional characterization of directed T7 RNAP mutants to both reveal the architecture of the duplex DNA that flanks the transcription bubble in the T7 RNAP initiation and elongation complexes, and to define the function of the interactions made by these duplex elements. We find that downstream duplex interactions made with a cluster of lysines (K711/K713/K714) are present during both elongation and initiation where they contribute to stabilizing a bend in the downstream DNA that is important for promoter opening. The upstream DNA in the elongation complex is also found to be sharply bent at the upstream edge of the transcription bubble, thereby allowing formation of upstream duplex:polymerase interactions that contribute to elongation complex stability.

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Structure of a T7 RNA polymerase elongation complex at 2.9 Å resolution

Cited by 226 publications

References 49 publications

Sequential multiple functions of the conserved sequence in sequence-specific termination by T7 RNA polymerase

Sequential multiple functions of the conserved sequence in sequence-specific termination by T7 RNA polymerase

The φ29 DNA polymerase:protein-primer structure suggests a model for the initiation to elongation transition

Functional Architecture of T7 RNA Polymerase Transcription Complexes

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