DNA bending in transcription initiation

Electrophoretic mobility shift (bandshift) phasing analysis and rotational variant topological analysis were performed on initiation complexes formed on the bacteriophage lambda PR promoter. Both the open complex and an abortive complex containing a short RNA primer extending to +3 were characterized. The two methods were used to analyze a series of constructs containing tandemly repeated copies of the PR promoter, with the repeat length increased in single base pair increments to progressively change the rotational setting of adjacent copies. The phasing effect observed in bandshift analysis of open complexes formed on this set of constructs provided qualitative evidence for the presence of a bend. Subsequent rotational variant topological analysis confirmed this and quantified the overall bend angle in the open complex as well as in the +3 abortive complex: a bend of 49 degrees +/- 7 degrees was measured for the open complex, while a bend of 47 degrees +/- 11 degrees was measured for the +3 complex, i.e., the two bends are the same. However, the topological results are not consistent with extensive superhelical wrapping of DNA on either complex as has been proposed. The two complexes do differ in the size of the transcription bubble: the open complex contains a 10.4 +/- 0.1 bp bubble, while that of the +3 complex is 12.2 +/- 0.1 bp, a result consistent with "DNA scrunching" during the onset of transcription. A model for the overall path of the DNA in the open complex is presented that is consistent with the measured bend angle. Measurement of both bubble size and overall bend angle complements the results of crystal structures in providing an enhanced description of the solution structures of the intact initiation complexes.