Related Experiment Videos
The +2 NTP binding drives open complex formation in T7 RNA polymerase
Natalie M Stano1, Mikhail K Levin, Smita S Patel
1Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
The Journal of Biological Chemistry
|August 2, 2002
Summary
Initiating NTPs are crucial for T7 RNA polymerase transcription. The +2 nucleotide triphosphate, correctly base-paired, drives open complex formation, with subsequent binding of the +1 NTP showing cooperativity.
Area of Science:
- Molecular Biology
- Biochemistry
- Enzymology
Background:
- Transcription initiation by T7 RNA polymerase involves promoter binding, strand separation, nucleotide binding, and phosphodiester bond synthesis.
- Promoter opening is rapid but incomplete without initiating NTPs.
Purpose of the Study:
- To investigate the impact of initiating NTPs on the kinetics and thermodynamics of open complex formation.
- To elucidate the role of specific NTPs in stabilizing the transcription pre-initiation complex.
Main Methods:
- Transient state kinetic studies were employed to analyze the formation of the open complex.
- The effects of different initiating NTPs (+1 and +2 positions) on complex stability were assessed.
Main Results:
- Open complex formation proceeds through an intermediate (ED(c) to ED(o)) with unfavorable equilibrium.
- The presence of a triphosphate NTP at the +2 position, correctly base-paired, drives near-complete open complex formation.
- Binding of the +2 NTP stabilizes the complex and facilitates subsequent +1 NTP binding, indicating cooperativity.
Conclusions:
- Initiating NTPs, particularly a triphosphate at the +2 position, are essential for stabilizing the open complex in T7 RNA polymerase transcription.
- The DNA template within the open complex is positioned to bind the +2 NTP, which in turn influences +1 NTP binding.
- Understanding these initiation steps provides insights into the regulation of transcription by NTP availability and promoter sequence elements.