Promoter clearance by T7 RNA polymerase. Initial bubble collapse and transcript dissociation monitored by base analog fluorescence

J Biol Chem. 2002 Jan 25;277(4):2725-31. doi: 10.1074/jbc.M108856200. Epub 2001 Nov 1.


Footprinting, fluorescence, and x-ray structural information from the initial, promoter-bound complex of T7 RNA polymerase describes the very beginning of the initiation of transcription, whereas recent fluorescence and biochemical studies paint a preliminary picture of an elongation complex. The current work focuses on the transition from an initially transcribing, promoter-bound complex to an elongation complex clear of the promoter. Fluorescence quenching is used to follow the melted state of the DNA bubble, and a novel approach using a locally mismatched fluorescent base analog reports on the local structure of the heteroduplex. Fluorescent base analogs placed at positions -2 and -1 of the promoter indicate that this initially melted, nontranscribed region remains melted as the polymerase translocates through to position +8. In progressing to position +9, this region of the DNA bubble begins to collapse. Probes placed at positions +1 and +2 of the template strand indicate that the 5' end of the RNA remains in a heteroduplex as the complex translocates to position +10. Subsequent translocation leads to sequential dissociation of the first 2 bases of the RNA. These results show that the initially transcribing complex bubble can reach a size of up to 13 base pairs and a maximal heteroduplex length of 10 base pairs. They further indicate that initial bubble collapse precedes dissociation of the 5' end of the RNA.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 2-Aminopurine / chemistry
  • Base Pair Mismatch
  • DNA / metabolism
  • DNA-Directed RNA Polymerases / genetics*
  • Fluorescent Dyes / pharmacology*
  • Models, Biological
  • Models, Genetic
  • Nucleic Acid Conformation
  • Promoter Regions, Genetic*
  • RNA / metabolism
  • RNA, Messenger / metabolism*
  • Spectrometry, Fluorescence
  • Transcription, Genetic*
  • Viral Proteins


  • Fluorescent Dyes
  • RNA, Messenger
  • Viral Proteins
  • 2-Aminopurine
  • RNA
  • DNA
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases