Pausing and termination by bacteriophage T7 RNA polymerase

Lyakhov, D. L.; He, Biao; Zhang, Xing; Studier, F. William; Dunn, John J.; McAllister, William T.

doi:10.1006/jmbi.1998.1854

Cited by 93 publications

(134 citation statements)

References 30 publications

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“…Consistent with previous results (14), termination by WT ECs at the PTH site was rapid, and Ϸ70% of the complexes terminated within 16 sec (Fig. 4B).…”

Section: Crosslinking the Rnap In The Ic Conformation Blocks Transcrisupporting

confidence: 92%

“…4B), a configuration that was first identified in a cloned copy of the human preparathyroid hormone gene (26). The downstream U-rich region appears to favor termination, and most transcription complexes that encounter the PTH signal rapidly terminate (14). By contrast, the class II signal found in the concatemer junction (CJ) of replicating T7 DNA (Fig.…”

Section: Crosslinking the Rnap In The Ic Conformation Blocks Transcrimentioning

confidence: 92%

“…By contrast, the class II signal found in the concatemer junction (CJ) of replicating T7 DNA (Fig. 4 A) lacks an extended U-rich region, and most transcription complexes that encounter this signal pause and then slowly resume transcription (14,16). The different responses of ECs at these two signals is likely to ref lect differences in stability of the paused complexes caused by the U-rich region, which is known to decrease the stability of an RNA-DNA hybrid (27).…”

Section: Crosslinking the Rnap In The Ic Conformation Blocks Transcrimentioning

confidence: 98%

“…This type of signal is similar to the intrinsic terminators used by bacterial RNAPs, and indeed, T7 RNAP has been found to use a number of such signals, suggesting a common mechanism of termination (9-11). Class II pause͞termination signals such as PTH, the termination site for T7 RNAP found in human preparathyroid hormone gene, do not involve an apparent RNA structure, but require a conserved 8-bp sequence [(C͞U)AUCUGU(U͞A)] encoded in the DNA template (11)(12)(13)(14)(15). Although the class II signal located in the concatamer junction (CJ) of T7 DNA serves as a strong pause site that does not give rise to efficient termination, the presence of additional U residues downstream of the conserved sequence results in efficient termination (13,14,16).…”

Section: Crosslink ͉ Transcriptionmentioning

confidence: 99%

“…Class II pause͞termination signals such as PTH, the termination site for T7 RNAP found in human preparathyroid hormone gene, do not involve an apparent RNA structure, but require a conserved 8-bp sequence [(C͞U)AUCUGU(U͞A)] encoded in the DNA template (11)(12)(13)(14)(15). Although the class II signal located in the concatamer junction (CJ) of T7 DNA serves as a strong pause site that does not give rise to efficient termination, the presence of additional U residues downstream of the conserved sequence results in efficient termination (13,14,16). Termination at class I and class II signals appears to involve separate mechanisms, as mutations of the RNAP that prevent termination at class II signals do not affect termination at class I signals (9,17,18).…”

Section: Crosslink ͉ Transcriptionmentioning

confidence: 99%

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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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“…Consistent with previous results (14), termination by WT ECs at the PTH site was rapid, and Ϸ70% of the complexes terminated within 16 sec (Fig. 4B).…”

Section: Crosslinking the Rnap In The Ic Conformation Blocks Transcrisupporting

confidence: 92%

Section: Crosslinking the Rnap In The Ic Conformation Blocks Transcrimentioning

confidence: 92%

Section: Crosslinking the Rnap In The Ic Conformation Blocks Transcrimentioning

confidence: 98%

Section: Crosslink ͉ Transcriptionmentioning

confidence: 99%

Section: Crosslink ͉ Transcriptionmentioning

confidence: 99%

See 3 more Smart Citations

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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Kinetic modeling and simulation of in vitro transcription by phage T7 RNA polymerase

Arnold

Siemann

Scharnweber

et al. 2001

Biotechnol. Bioeng.

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This study provides a mathematical model of T7 RNA polymerase (T7 RNAP) kinetics under in vitro conditions targeted at application of this model to simulation of dynamic transcription performance. A functional dependence of transcript synthesis rate is derived based on: (a) essential reactant concentrations, including T7 RNAP and its promoter, substrate nucleotides, and the inhibitory byproduct inorganic pyrophosphate; (b) a distinction among vector characteristics such as recognition sequences regulating transcription initiation and termination, respectively; and (c) specific properties of the nucleotide sequence including both transcript length and nucleotide composition. Inactivation kinetics showed a half‐life of T7 RNAP activity of 50 min under the conditions applied in vitro using the isolated enzyme. Model parameters and their precision are estimated using dynamic simulation and nonlinear regression analysis. The particular novelty of this model is its capability to incorporate linear genomic sequence information for simulation of nonlinear in vitro transcription kinetics. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 548–561, 2001.

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Specific labeling: An effective tool to explore the RNA world

2015

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Our knowledge about the functional diversity and importance of RNA in biology has grown enormously over the past three decades and has driven efforts to develop better tools to characterize RNAs. Amongst these tools are methods for preparing specifically labeled or chemically modified RNAs, which are essential for basic research, biomedical, and clinical applications. Understanding the potential and limits of these different RNA synthesis and labeling strategies is important in deciding how to approach the preparation of a particular RNA molecule. Here, we review these various labeling methods and future directions of the field.

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Pausing and termination by bacteriophage T7 RNA polymerase

Cited by 93 publications

References 30 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

Kinetic modeling and simulation of in vitro transcription by phage T7 RNA polymerase

Specific labeling: An effective tool to explore the RNA world

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