Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

54-POS Board 54 DNA WRAPPING IN OPEN TRANSCRIPTION INITIATION COMPLEXES IS THE MAJOR COMPONENT OF THEIR STABILITY Robert P Sosa 1,2,3 ; Alfredo J Florez-Ariza 4,5 ; Bibiana G Onoa 2,7 ; Cesar Diaz-Celiz 2,7 ; Alexandre Cassago 4 ; Paulo S de Oliveira 4 ; Rodrigo V Portugal 4,5 ; Daniel G Guerra 3 ; Carlos J Bustamante 6,7,8 ; 1 UC Berkeley, Biophysics, berkeley, CA, USA 2 Quantitative Biosciences-QB3, berkeley, CA, USA 3 Universidad Peruana Cayetano Heredia (UPCH), Ciencias y Filosofia, Lima, Peru 4 Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, Brazil 5 Universidade de Sao Paulo, Fisica, Sao carlos, Brazil 6 UC Berkeley, Molecular and Cell Biology, Chemistry and Physics, berkeley, CA, USA The interaction of RNA polymerase (RNAP) and a DNA promoter leads to the formation of the open transcription initiation complex (RPo), the major regulatory checkpoint in transcription. DNA wrapping is a common process in RPo formation and despite its potential importance, wrapping has been problematic to study due to the difficulty to separate the initial binding from the ensuing fast wrapping through techniques such as footprinting, fluorescence, and AFM. We investigate wrapping in RPo using optical tweezers and determine its structure using transmission electron microscopy and single particle analysis. Our results indicate that wrapping is reversible, cooperative, and spontaneous, with a change in free energy similar to the change in free energy of RPo formation (~ -13 kcal/mol), indicating that this process is the principal contributor to the complex’s stability. The RPo structure at 17 Å resolution reveals that wrapping encompasses position -76 to +18 along the promoter, with an angle of ~245°, and that RNAP alpha subunit C-terminal domains (aCTDs) interact with the proximal and middle upstream promoter sequences. Also, ~80% of the wrapping energy is contributed by aCTDs in interaction with non-specific upstream DNA sequences and we show that the transcriptional modulator ppGpp reduces the extent and strength of wrapping. Finally, we demonstrate a correlation between wrapping-dependent stability and RNAP promoter escape as well as RPo formation. 7 Howard Hughes Medical Institute, berkeley, CA, USA 8 Kavli Energy Nanosciences Institute, berkeley, CA, USA

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