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 language | English (US) |
|---|---|
| Pages (from-to) | 37292-37300 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 277 |
| Issue number | 40 |
| DOIs | |
| State | Published - Oct 4 2002 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
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 journal › Article
}
TY - JOUR
T1 - The +2 NTP binding drives open complex formation in T7 RNA polymerase
AU - Polymerase, R. N A
AU - Stano, Natalie M.
AU - Levin, Mikhail K.
AU - Patel, Smita S.
PY - 2002/10/4
Y1 - 2002/10/4
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0037020232&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037020232&partnerID=8YFLogxK
U2 - 10.1074/jbc.M201600200
DO - 10.1074/jbc.M201600200
M3 - Article
C2 - 12151383
AN - SCOPUS:0037020232
VL - 277
SP - 37292
EP - 37300
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 40
ER -