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Related Experiment Videos

Sequential multiple functions of the conserved sequence in sequence-specific termination by T7 RNA polymerase.

Younghee Sohn1, Changwon Kang

  • 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea.

Proceedings of the National Academy of Sciences of the United States of America
|December 24, 2004
PubMed
Summary

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The conserved sequence (CS) in E. coli rrnB terminator t1 plays a crucial role in transcription termination. It sequentially interacts with RNA polymerase, facilitating termination by influencing polymerase-RNA binding and DNA duplex reformation.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Escherichia coli rrnB terminator t1 features both hairpin-dependent (class I) and sequence-specific (class II) termination signals.
  • The class II signal comprises a conserved sequence (CS) and a T-rich region.

Purpose of the Study:

  • To investigate the conformational changes of T7 RNA polymerase during interaction with the class II termination signal.
  • To elucidate the role of the conserved sequence (CS) in transcription termination.

Main Methods:

  • Stepwise elongation of T7 RNA polymerase on immobilized DNA templates lacking the class I signal.
  • Analysis of polymerase-RNA and polymerase-DNA interactions using radiolabeled transcript retention and cross-linking assays.

Main Results:

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  • Polymerase stability decreases upon reaching the CS-DNA duplex.
  • Distinct changes in polymerase-RNA interaction occur as elongation progresses through the CS.
  • Reformation of the CS-DNA duplex and altered transcription bubble dynamics are observed before termination.

Conclusions:

  • The CS duplex is essential for sequential binding to RNA polymerase both upstream and downstream of the RNA-DNA heteroduplex.
  • These interactions facilitate transcription termination at the rrnB t1 terminator.