The +2 NTP binding drives open complex formation in T7 RNA polymerase

R. N A Polymerase, Natalie M. Stano, Mikhail K. Levin, Smita S. Patel

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Transcription initiation as catalyzed by T7 RNA polymerase consists primarily of promoter binding, strand separation, nucleotide binding, and synthesis of the first phosphodiester bond. The promoter strand separation process occurs at a very fast rate, but promoter opening is incomplete in the absence of the initiating NTPs. In this paper, we investigate how initiating NTPs affect the kinetics and thermodynamics of open complex formation. Transient state kinetic studies show that the open complex, EDo, is formed via an intermediate EDc, and the conversion of EDc to EDo occurs with an unfavorable equilibrium constant. In the presence of the initiating NTP that base-pairs with the template at position +2, the process of open complex formation is nearly complete. Our studies reveal that the nucleotide that drives open complex formation needs to be a triphosphate and to be correctly base-paired with the template. These results indicate that the melted template DNA in the open complex is positioned to bind the +2 NTP. The addition of +1 NTP alone does not stabilize the open complex; nor is it required for +2 NTP binding. However, there appears to be cooperativity in initiating NTP binding in that the binding of +2 NTP facilitates +1 NTP binding. The dissection of the initiation pathway provides insights into how open complex formation steps that are sensitive to the promoter sequence upstream from the initiation start site modulate the affinity of initiating NTPs and allow transcription initiation to be regulated by initiating NTP concentration.

Original languageEnglish (US)
Pages (from-to)37292-37300
Number of pages9
JournalJournal of Biological Chemistry
Volume277
Issue number40
DOIs
StatePublished - Oct 4 2002

Fingerprint

Transcription
Nucleotides
Dissection
Kinetics
Equilibrium constants
Thermodynamics
Base Pairing
DNA
bacteriophage T7 RNA polymerase
triphosphoric acid

ASJC Scopus subject areas

  • Biochemistry

Cite this

Polymerase, R. N. A., Stano, N. M., Levin, M. K., & Patel, S. S. (2002). The +2 NTP binding drives open complex formation in T7 RNA polymerase. Journal of Biological Chemistry, 277(40), 37292-37300. https://doi.org/10.1074/jbc.M201600200

The +2 NTP binding drives open complex formation in T7 RNA polymerase. / Polymerase, R. N A; Stano, Natalie M.; Levin, Mikhail K.; Patel, Smita S.

In: Journal of Biological Chemistry, Vol. 277, No. 40, 04.10.2002, p. 37292-37300.

Research output: Contribution to journalArticle

Polymerase, RNA, Stano, NM, Levin, MK & Patel, SS 2002, 'The +2 NTP binding drives open complex formation in T7 RNA polymerase', Journal of Biological Chemistry, vol. 277, no. 40, pp. 37292-37300. https://doi.org/10.1074/jbc.M201600200
Polymerase, R. N A ; Stano, Natalie M. ; Levin, Mikhail K. ; Patel, Smita S. / The +2 NTP binding drives open complex formation in T7 RNA polymerase. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 40. pp. 37292-37300.
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