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Related Experiment Videos

T7 RNA polymerase elongation complex structure and movement.

J Huang1, R Sousa

  • 1Department of Biochemistry, University of Texas Health Sciences Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7760, USA.

Journal of Molecular Biology
|October 14, 2000
PubMed
Summary

This study reveals how T7 RNA polymerase (RNAP) moves on DNA during transcription. NTP binding stabilizes the enzyme

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Bacterial RNA polymerase (RNAP) is crucial for gene expression.
  • Understanding RNAP's translocation mechanism is key to controlling transcription.

Purpose of the Study:

  • To characterize the structure and dynamics of T7 RNAP elongation complexes (ECs).
  • To investigate the role of nucleotide binding in RNAP translocation and positioning.

Main Methods:

  • Enzyme digestion (exonuclease III, lambda exonuclease, RNAse T1).
  • Chemical probing (KMnO4).
  • Analysis of halted transcription elongation complexes (ECs).

Main Results:

  • The transcription bubble is ~9 bases, with a 7-8 bp RNA:DNA hybrid.
  • NTP binding causes downstream translocation and restricts RNAP mobility.
  • RNAP can slide upstream in the absence of NTPs, stabilizing post-translocated position upon binding.

Conclusions:

  • NTP binding is essential for stabilizing RNAP in the correct post-translocated position.
  • RNAP translocation is regulated by NTP binding and potential conformational changes.

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