DNA bubble formation in transcription initiation

The properties of the DNA bubble in the transcription open complex have been characterized by topological analysis of DNA circles containing the lac UV5 promoter or the PR promoter from bacteriophage lambda. Topological analysis is particularly well suited to this purpose since it quantifies the changes in DNA duplex geometry caused by bubble formation as well as by superhelical DNA wrapping. The duplex unwinding that results from bubble formation is detected as a reduction in topological linking number of the DNA circle, and the precision of this measurement has been enhanced in the current study through the use of 8 or 10 promoter copies per circle. Several lines of evidence indicate that the linking number change induced by open complex formation is essentially all due to bubble generation, with very little derived from superhelical wrapping. Accordingly, the linking number change of -1.17 measured for the lac UV5 promoter indicates that the size of the lac UV5 bubble is about 12.3 base pairs, while the change of -0.98 measured for the lambda PR promoter indicates that the lambda PR bubble is 10.3 base pairs. It was also found that the presence or absence of magnesium ion had little effect on the value of the linking number change, a result that resolves the uncertainty associated with use of chemical probes to study the effect of magnesium on bubble size. Finally, the magnitude of linking number change increases progressively when the 3' end of a transcript is extended to +2 and +3 in an abortive initiation complex. This indicates that the transcription bubble expands at its leading edge in the abortive complex, results that confirm and extend the proposal of a DNA "scrunching" mechanism at the onset of transcription. These results are relevant to several models for the structure of DNA in the functional open complex in solution, and provide an important complement to the structural information available from recent crystal structures.