DNA melting by RNA polymerase at the T7A1 promoter precedes the rate-limiting step at 378C and results in the accumulation of an off-pathway intermediate

Anastasia Rogozina (1) , Evgeny Zaychikov (1) , Malcolm Buckle (2) , Hermann Heumann (1) , Bianca Sclavi (2)

1 MPIB - Max-Planck-Institut für Biochemie = Max Planck Institute of Biochemistry
2 LBPA - Laboratoire de Biologie et de Pharmacologie Appliquée

The formation of a transcriptionally active complex by RNA polymerase involves a series of short-lived structural intermediates where protein conforma-tional changes are coupled to DNA wrapping and melting. We have used time-resolved KMnO 4 and hydroxyl-radical X-ray footprinting to directly probe conformational signatures of these complexes at the T7A1 promoter. Here we demonstrate that DNA melting from m12 to m4 precedes the rate-limiting step in the pathway and takes place prior to the formation of full downstream contacts. In addition, on the wild-type promoter, we can detect the accumulation of a stable off-pathway intermediate that results from the absence of sequence-specific contacts with the melted non-consensus-10 region. Finally, the comparison of the results obtained at 378C with those at 208C reveals significant differences in the structure of the intermediates resulting in a different pathway for the formation of a transcriptionally active complex.

Domaines: Sciences du Vivant [q-bio]; Biochimie, Biologie Moléculaire

Anastasia Rogozina, Evgeny Zaychikov, Malcolm Buckle, Hermann Heumann, Bianca Sclavi. DNA melting by RNA polymerase at the T7A1 promoter precedes the rate-limiting step at 378C and results in the accumulation of an off-pathway intermediate. Nucleic Acids Research, 2009, pp.1 - 15. ⟨10.1093/nar/gkp560⟩. ⟨hal-02374037⟩