Strand separation required for initiation of replication at the chromosomal origin of E.coli is facilitated by a distant RNA--DNA hybrid.

The Escherichia coli initiator protein, dnaA complexed with ATP, binds to the replication origin (oriC) and melts the duplex in the AT-rich part of oriC. Opening of the duplex requires a minimum temperature and superhelix density. A low level of HU protein (5 molecules/oriC) is stimulatory, probably by facilitating DNA bending. High levels of HU protein (136 molecules/oriC) restrain the free superhelicity. Under adverse conditions for oriC melting (i.e. absence of HU, high levels of HU, low temperature or low superhelicity), transcription can activate initiation of replication by generating an R-loop located even a large distance from oriC. The strand-separation stage at oriC prior to the entry of the dnaB helicase was specifically stimulated. Activation by the R-loop was blocked when a stretch of 14 GC base pairs was inserted between the loop and oriC. Thus, activation of oriC by the distantly located R-loop appears to require propagation of DNA melting through the intervening sequence.