The Transition to an Elongation Complex by T7 RNA Polymerase Is a Multistep Process

Bandwar, Rajiv P.; Ma, Na; Emanuel, Steven; Anikin, Michael; Vassylyev, Dmitry G.; Patel, Smita S.; McAllister, William T.

doi:10.1074/jbc.m702589200

Cited by 25 publications

(38 citation statements)

References 33 publications

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“…Halting at positions ϩ8, ϩ9, and ϩ10 shows no evidence of a transition to elongation, but the transition is observed in complexes halted at position ϩ11 and beyond. In contrast to earlier proposals (19), the P266L mutant is transitioning to elongation later (at longer RNA lengths) than wild type.…”

Section: Resultscontrasting

confidence: 49%

“…3 describes the possible outcomes available to an initially transcribing RNA polymerase. Recent studies have shown that the transition to elongation does not occur at a discrete RNA length, but rather begins with very low probability at position ϩ8, increasing in probability as the complex translocates to longer RNA lengths (18,19). To characterize kinetics at a specific position, we can halt transcription by leaving out a specific NTP from the mix.…”

Section: Resultsmentioning

confidence: 99%

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New Insights into the Mechanism of Initial Transcription

Ramírez-Tapia

Martin

2012

Journal of Biological Chemistry

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Background: RNA polymerases must couple the energetics of nucleotide addition to the timed release of promoter contacts. Results: A mutant that aborts less during initial transcription releases the promoter at longer RNA lengths. Conclusion: Hybrid growth remodels the protein to disrupt promoter contacts; the protein, in turn, pushes back on the hybrid, leading to abortive instability. Significance: The model extends to multisubunit RNA polymerases.

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Section: Resultscontrasting

confidence: 49%

Section: Resultsmentioning

confidence: 99%

New Insights into the Mechanism of Initial Transcription

Ramírez-Tapia

Martin

2012

Journal of Biological Chemistry

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“…We also obtained a reasonable fit of the C-terminal domain of Rpo41 to the elongation conformation of T7 RNAP (supplemental Fig. S9), suggesting that perhaps Rpo41 undergoes protein conformational changes during transition from initiation to elongation analogous to T7 RNAP (46,47).…”

Section: Discussionmentioning

confidence: 92%

Mitochondrial Transcription Factor Mtf1 Traps the Unwound Non-template Strand to Facilitate Open Complex Formation

Paratkar¹,

Patel²

2010

Journal of Biological Chemistry

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The catalytic subunit of the mitochondrial (mt) RNA polymerase (RNAP) is highly homologous to the bacteriophage T7/T3 RNAP. Unlike the phage RNAP, however, the mtRNAP relies on accessory proteins to initiate promoterspecific transcription. Rpo41, the catalytic subunit of the Saccharomyces cerevisiae mtRNAP, requires Mtf1 for opening the duplex promoter. To elucidate the role of Mtf1 in promoter-specific DNA opening, we have mapped the structural organization of the mtRNAP using site-specific protein-DNA photo-cross-linking studies. Both Mtf1 and Rpo41 cross-linked to distinct sites on the promoter DNA, but the dominant cross-links were those of the Mtf1, which indicates a direct role of Mtf1 in promoter-specific binding and initiation. Strikingly, Mtf1 cross-linked with a high efficiency to the melted region of the promoter DNA, based on which we suggest that Mtf1 facilitates DNA melting by trapping the non-template strand in the unwound conformation. Additional strong cross-links of the Mtf1 were observed with the ؊8 to ؊10 base-paired region of the promoter. The cross-linking results were incorporated into a structural model of the mtRNAP-DNA, created from a homology model of the C-terminal domain of Rpo41 and the available structure of Mtf1. The promoter DNA is sandwiched between Mtf1 and Rpo41 in the structural model, and Mtf1 closely associates mainly with one face of the promoter across the entire nona-nucleotide consensus sequence. Overall, the studies reveal that in many ways the role of Mtf1 is analogous to the transcription factors of the multisubunit RNAPs, which provides an intriguing link between single-and multisubunit RNAPs. The mitochondrial (mt)2 RNA polymerase (RNAP) is closely related to the single-subunit bacteriophage T7/T3 RNAP (1). Their C-terminal ϳ800 amino acids show ϳ30% sequence identity to T7 RNAP (1-3). Despite this similarity, the mtRNAP depends on transcription factors for promoter-specific initiation (4 -7). The core RNAP subunit of the Saccharomyces cerevisiae, Rpo41, requires one major factor, Mtf1 (or sc-MtfB) (6, 8 -11), whereas the human core mtRNAP requires two factors, mtTFB1/2 and mtTFA (12-17).Rpo41 by itself does not recognize and melt the mt promoter or initiate RNA synthesis unless the promoter is premelted around the transcription site (18 -20). Similarly, there is no evidence that Mtf1 by itself interacts with the promoter (7, 18). When Mtf1 and Rpo41 are present together, the complex (21) melts the promoter from Ϫ4 to ϩ2 without requiring initiating NTPs (18). Based on these results, it has been suggested that Rpo41 lacks the mechanism for melting/stabilizing the open promoter and relies on Mtf1 for promoter-specific binding, melting, and stabilizing of the melted promoter.There are two general ways in which Mtf1 can facilitate promoter opening (Fig. 1). In Model A, only the Rpo41 protein within the mtRNAP complex (Rpo41⅐Mtf1) interacts with the promoter DNA, and Mtf1 acts allosterically. Through protein-protein interactions, Mtf1 causes conformational changes with...

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“…During the purification of T7 RNAP, it was found that the protein can be nicked between amino acids 179 and 180, located in the H-loop domain (25,26). After T7 RNAP binds to its promoter, the H-loop domain is known to refold during the enzyme's transition from an initiation complex into an elongation complex (25,27). The C-terminal fragment of nicked T7 RNAP (amino acids 180-880) was found to bind P T7 on its own but was unable to synthesize full-length mRNA (25).…”

Section: Resultsmentioning

confidence: 99%

Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants

Shis

Bennett

2013

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

136

147

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The construction of synthetic gene circuits relies on our ability to engineer regulatory architectures that are orthogonal to the host's native regulatory pathways. However, as synthetic gene circuits become larger and more complicated, we are limited by the small number of parts, especially transcription factors, that work well in the context of the circuit. The current repertoire of transcription factors consists of a limited selection of activators and repressors, making the implementation of transcriptional logic a complicated and component-intensive process. To address this, we modified bacteriophage T7 RNA polymerase (T7 RNAP) to create a library of transcriptional AND gates for use in Escherichia coli by first splitting the protein and then mutating the DNA recognition domain of the C-terminal fragment to alter its promoter specificity. We first demonstrate that split T7 RNAP is active in vivo and compare it with fulllength enzyme. We then create a library of mutant split T7 RNAPs that have a range of activities when used in combination with a complimentary set of altered T7-specific promoters. Finally, we assay the two-input function of both wild-type and mutant split T7 RNAPs and find that regulated expression of the N-and C-terminal fragments of the split T7 RNAPs creates AND logic in each case. This work demonstrates that mutant split T7 RNAP can be used as a transcriptional AND gate and introduces a unique library of components for use in synthetic gene circuits.protein fragment complementation | H-loop S ynthetic gene circuits provide valuable insights into biophysical phenomena by enabling the construction and characterization of genetic systems from the ground up (1-3). Further, synthetic gene circuits are rapidly becoming core components of biotechnologies in metabolic engineering and in medicine (4-7). The continued development of synthetic gene circuits calls for the ability to construct larger and more complex circuits, which in turn necessitates the development of additional parts and component libraries with which to build them (8, 9).Recent efforts to address the "component problem" (10) (ie, the lack of well-characterized orthogonal parts with which to build synthetic gene circuits) have led to the development of novel transcriptional and translational regulators. The effort to develop transcriptional regulators has, for instance, involved transplanting transcriptional regulators from genetically distant microorganisms into a particular chassis organism such as Escherichia coli. This reimagining of transcriptional regulatory systems has led to novel ligand-sensitive transcription factor-promoter pairs (11) and transcriptional logic gates (12). In addition, recent work has made it possible to synthetically regulate protein translation through, for example, modulating access to the ribosome binding site (RBS) or varying the stability of the transcript (5, 13-16).At the core of many synthetic gene circuits lie transcriptional logic gates. Transcriptional logic gates mirror digital logic gates...

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The Transition to an Elongation Complex by T7 RNA Polymerase Is a Multistep Process

Cited by 25 publications

References 33 publications

New Insights into the Mechanism of Initial Transcription

New Insights into the Mechanism of Initial Transcription

Mitochondrial Transcription Factor Mtf1 Traps the Unwound Non-template Strand to Facilitate Open Complex Formation

Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants

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