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In Vitro Transcription Assays and Their Application in Drug Discovery
Published on: September 20, 2016
Structural biophysics of the NusB:NusE antitermination complex.
Ranabir Das1, Sandra Loss, Jess Li
1Structural Biophysics Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
Journal of Molecular Biology
|January 8, 2008
Summary
Host factors NusB and NusE form a complex that enhances RNA binding, promoting antitermination in prokaryotic transcription. This interaction stabilizes the transcription complex, allowing RNA polymerase to bypass termination sites.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Prokaryotic transcription relies on host factors interacting with RNA polymerase and mRNA.
- Antitermination involves NusB and NusE forming a heterodimer that binds boxA RNA.
- This complex stabilizes the transcription machinery, preventing premature termination.
Purpose of the Study:
- To elucidate the molecular mechanism of NusB-NusE interaction in antitermination.
- To determine the structure of NusB from Aquifex aeolicus.
- To characterize the binding of NusB and NusE and identify their interaction sites.
Main Methods:
- NMR spectroscopy
- Isothermal titration calorimetry
- Gel filtration
- Analytical ultracentrifugation
- NMR chemical shift perturbation studies
Main Results:
- Determined the structure of Aquifex aeolicus NusB.
- Characterized the NusB-NusE binding interaction.
- Identified the NusE binding site on NusB and in the ternary complex.
- Observed a specific loop in NusB (residues 113-118) affected by NusE binding in the ternary complex.
Conclusions:
- NusE enhances NusB's affinity for boxA RNA, crucial for efficient antitermination.
- The NusB-NusE interaction site is conserved between binary and ternary complexes.
- Structural insights into the NusB/NusE/boxA RNA complex provide a basis for understanding antitermination regulation.
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