Monday, 23 May 2005 - 3:50 PM
286

This presentation is part of: Gene Expression: Transcription

Direct Observation of Abortive Initiation and Promoter Escape: Single-Molecule DNA Nanomanipulation

Andrei Revyakin1, Chenyu Liu1, Terence Strick2, and Richard H. Ebright1. (1) Howard Hughes Medical Institute, Rutgers University, Piscataway, NJ, (2) Institut Jacques Monod, Paris, France

DNA-footprinting results indicate that the upstream boundary of the DNA segment protected by RNA polymerase (RNAP) is identical in RNAP-promoter open complexes and in RNAP-promoter complexes engaged in abortive synthesis of RNA products up to 9-10 nt in length. To account for the ability of RNAP to synthesize RNA products up to 9-10 nt in length without apparent change in the upstream boundary of the protected region, three models have been proposed: (i) "inchworming" which invokes transient expansion of RNAP, (ii) "scrunching," which invokes transient compaction of DNA, and (iii) "transient excursions," which invokes transient cycles of forward and reverse RNAP translocation.

We have used DNA nanomanipulation to define the extent of promoter unwinding, at the single-molecule level, in RNAP-promoter open complexes and in RNAP-promoter complexes engaged in iterative abortive synthesis. The results show unequivocally that the extent of promoter unwinding increases during abortive synthesis, increasing by a factor of up to ~1.5-fold in a manner dependent on RNA-product length.

We further have used DNA nanomanipulation to monitor the extent of promoter unwinding, at the single-molecule level, in real time, during promoter escape. The results show unequivocally that the extent of promoter unwinding transiently increases during promoter escape, increasing by a factor of ~1.5 during promoter escape, then returning to the original level during elongation.


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