Sequential Release of Promoter Contacts during Transcription Initiation to Elongation Transition

Bandwar, Rajiv P.; Tang, Guiqian; Patel, Smita S.

doi:10.1016/j.jmb.2006.05.029

Cited by 23 publications

(60 citation statements)

References 47 publications

(107 reference statements)

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“…By ϩ12 and at further positions, only a single population at the low-FRET value was observed. The new low-FRET species appeared after 8-nt synthesis when the transition from IC to EC was observed from other studies of T7 RNAP (28)(29)(30)(31). Therefore, the low-FRET population was assigned as the EC state.…”

mentioning

confidence: 53%

“…The sudden increase in RNA polymerization rate after 8-nt synthesis coincides with the stage when the specificity loop loses its interactions with the promoter (28,36). Loss-of-specificity loop interactions could increase the rate of RNA synthesis by removing the barrier for the rotational conformational change, facilitating further elongation of the RNA from 8 nt until EC formation.…”

Section: Kinetics Of Ic-to-ec Transition By Stopped-flow Fret Measurementioning

confidence: 97%

“…There is a Ϸ10-fold increase in rate from 8 to 9, Ϸ5-fold increase from 9 to 10, Ϸ7-fold increase from 11 to 12, and a cumulative 300-to 600-fold increase in the transition rate as the RNA extends from 8 to 12 nt. During the IC-to-EC transition, the initially opened transcription bubble from Ϫ4 to ϩ2 of the promoter DNA recloses to regenerate the duplex region (initial bubble collapse) (28)(29)(30). The kinetics of initial bubble reannealing were monitored by following the decrease in the fluorescence of 2-aminopurine (2AP) placed at Ϫ4NT (28).…”

Section: Kinetics Of Ic-to-ec Transition By Stopped-flow Fret Measurementioning

confidence: 99%

“…To determine whether changes in the promoter sequence alter the efficiency and/or the location of the IC-to-EC transition, the stopped-flow FRET studies were carried out with an altered T7 promoter with base pair mutation A-15C in the upstream promoter region (28,33). The late-phase FRET decrease indicative of the IC-to-EC transition was detectable in the A-15C promoter as early as 7-nt synthesis and was most efficient after 9-nt synthesis ( Fig.…”

Section: Kinetics Of Ic-to-ec Transition By Stopped-flow Fret Measurementioning

confidence: 99%

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Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase

Tang¹,

Roy²,

Bandwar³

et al. 2009

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

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The transition from initiation to elongation of the RNA polymerase (RNAP) is an important stage of transcription that often limits the production of the full-length RNA. Little is known about the RNAP transition kinetics and the steps that dictate the transition rate, because of the challenge in monitoring subpopulations of the transient and heterogeneous transcribing complexes in rapid and real time. Here, we have dissected the complete transcription initiation pathway of T7 RNAP by using kinetic modeling of RNA synthesis and by determining the initiation (IC) to elongation (EC) transition kinetics at each RNA polymerization step using single-molecule and stoppedflow FRET methods. We show that the conversion of IC to EC in T7 RNAP consensus promoter occurs only after 8-to 12-nt synthesis, and the 12-nt synthesis represents a critical juncture in the transcriptional initiation pathway when EC formation is most efficient. We show that the slow steps of transcription initiation, including DNA scrunching/ RNAP-promoter rotational changes during 5-to 8-nt synthesis, not the major conformational changes, dictate the overall rate of EC formation in T7 RNAP and represent key steps that regulate the synthesis of full-length RNA.abortive synthesis ͉ FRET ͉ rate-limiting ͉ T7 RNA polymerase ͉ transcription transition T he recruitment of RNA polymerase (RNAP) to the promoter and isomerization to a competent open complex are important regulatory steps in gene transcription (1, 2). Similarly, the transition from abortive initiation to processive elongation is an essential event of transcription that often dictates the rate at which the full-length RNA is made (3-9). During the abortive initiation phase, the RNAP maintains contacts with the promoter and catalyzes RNA polymerization by scrunching the template DNA (10-13). After a certain length of RNA is made, the RNAP switches transcription from promoter-specific to promoter-independent and processive RNA synthesis. Single-subunit and multisubunit RNAPs switch from the initiation complex (IC) to the elongation complex (EC) by undergoing specific structural rearrangements. For example, the single-subunit T7 RNAP protein undergoes major refolding of its N-terminal domain during EC transition that releases promoter contacts and results in the formation of RNA channel (14,15). The bacterial RNAP makes this transition by releasing the sigma factor from the promoter (7,(16)(17)(18)(19), and in eukaryotic RNAP II, this event is marked by the removal of several transcription factors, including TFIIB, TFIIE, TFIIF, and TFIIH (20). Although the initiation stage and the transition from IC to EC are major points of regulation that ultimately control gene expression, the rate-limiting steps governing these multistep processes and the kinetics of transition have not been elucidated.The challenge in studying the kinetic pathway of initiation and the IC-to-EC transition has been the transient and heterogeneous nature of the initially transcribing complexes. Studies of the transient heterogen...

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mentioning

confidence: 53%

Section: Kinetics Of Ic-to-ec Transition By Stopped-flow Fret Measurementioning

confidence: 97%

Section: Kinetics Of Ic-to-ec Transition By Stopped-flow Fret Measurementioning

confidence: 99%

Section: Kinetics Of Ic-to-ec Transition By Stopped-flow Fret Measurementioning

confidence: 99%

See 2 more Smart Citations

Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase

Tang¹,

Roy²,

Bandwar³

et al. 2009

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

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“…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%

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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Fluorescent Methods to Study Transcription Initiation and Transition into Elongation

Deshpande

Sultana

Patel

2014

Experientia Supplementum

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The DNA-dependent RNA polymerases induce specific conformational changes in the promoter DNA during transcription initiation. Fluorescence spectroscopy sensitively monitors these DNA conformational changes in real time and at equilibrium providing powerful ways to estimate interactions in transcriptional complexes and to assess how transcription is regulated by the promoter DNA sequence, transcription factors, and small ligands. Ensemble fluorescence methods described here probe the individual steps of promoter binding, bending, opening, and transition into the elongation using T7 phage and mitochondrial transcriptional systems as examples.

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Sequential Release of Promoter Contacts during Transcription Initiation to Elongation Transition

Cited by 23 publications

References 47 publications

Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase

Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase

New Insights into the Mechanism of Initial Transcription

Fluorescent Methods to Study Transcription Initiation and Transition into Elongation

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