Poster Presentation 16th Asian Conference on Transcription 2019

High-energy initiation complex retaining σ70 identified as complex hypersensitive to pyrophosphate (1127)

Nobuo Shimamoto 1 2 , Masahiko Imashimizu 1 3
  1. National Institute of Genetics, Japan, Kyoto, JAPAN, Japan
  2. Veritas Kitayama, Kyoto, Japan
  3. Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo, JAPAN

During transition from initiation to elongation in E. coli transcription, σ70is supposed to be released from transcription complex to remove a steric clash of the 5’ end of nascent RNA with the σ70regions 4 and 3.2 loop. The existence of such high-energy complex has been proposed in abortive initiationbut which does not lead to productive elongation.

In this study, we have found a high-energy transcription complex retaining σ70and 9 nt nascent RNA, that is long enough to reach σ70 region 4. It catalyzes the reverse reaction of elongation, pyrophosphorolysis, in the presence of low concentrations of PPi which does not inhibit elongation by most transcription complexes. We also demonstrated that the hypersensitivity to PPi was attributed to the complex performing productive RNA synthesis but not abortive RNA synthesis. The high-energy complex is only formed through the transcription initiation from a promoter but not from the reconstitution with RNA polymerase, DNA, and 9nt RNA, consistent with the interpretation that the energy originates from the breakage of β-γphosphodiester bond of NTP. These results provide energetic evidence for a high-energy initiation complex retaining σ70during the initiation-to-elongation transition. Cellular PPi as well as the high-energy conformation may accelerate the transition and the consequent productive elongation.

Imashimizu M. et al., JMB (2019) 431, 2528-42