Intrinsic translocation barrier as an initial step in pausing by RNA polymerase II

J Mol Biol. 2013 Feb 22;425(4):697-712. doi: 10.1016/j.jmb.2012.12.002. Epub 2012 Dec 10.


Pausing of RNA polymerase II (RNAP II) by backtracking on DNA is a major regulatory mechanism in control of eukaryotic transcription. Backtracking occurs by extrusion of the 3' end of the RNA from the active center after bond formation and before translocation of RNAP II on DNA. In several documented cases, backtracking requires a special signal such as A/T-rich sequences forming an unstable RNA-DNA hybrid in the elongation complex. However, other sequence-dependent backtracking signals and conformations of RNAP II leading to backtracking remain unknown. Here, we demonstrate with S. cerevisiae RNAP II that a cleavage-deficient elongation factor TFIIS (TFIIS(AA)) enhances backtracked pauses during regular transcription. This is due to increased efficiency of formation of an intermediate that leads to backtracking. This intermediate may involve misalignment at the 3' end of the nascent RNA in the active center of the yeast RNAP II, and TFIIS(AA) promotes formation of this intermediate at the DNA sequences, presenting a high-energy barrier to translocation. We proposed a three-step mechanism for RNAP II pausing in which a prolonged dwell time in the pre-translocated state increases the likelihood of the 3' RNA end misalignment facilitating a backtrack pausing. These results demonstrate an important role of the intrinsic blocks to forward translocation in pausing by RNAP II.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Base Sequence
  • DNA, Fungal / chemistry
  • DNA, Fungal / genetics
  • Kinetics
  • Models, Genetic
  • Mutation
  • Protein Transport
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism*
  • RNA, Fungal / genetics*
  • RNA, Fungal / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Transcription, Genetic*
  • Transcriptional Elongation Factors / genetics
  • Transcriptional Elongation Factors / metabolism


  • DNA, Fungal
  • RNA, Fungal
  • Saccharomyces cerevisiae Proteins
  • Transcriptional Elongation Factors
  • transcription factor S-II
  • RNA Polymerase II
  • RPB1 protein, S cerevisiae