Molecular mechanisms regulating the timing of initiation of DNA replication during the cell division cycle of Escherichia coli

In Escherichia coli, the frequency of initiation of chromosome replication determines the rate of cell division. The ability of E. coli to properly time the initiation of replication during the division cycle, thereby ensuring that each cell contains the appropriate number of chromosomes following division, is critical for normal cell growth. The molecular mechanisms which control the precise timing of initiation during the E. coli division cycle involve a host of both positive and negative factors which interact at the origin of replication, oriC. The purpose of this investigation was to examine the possible involvement of transcription in the control of initiation of chromosome replication and to identify the factors that define the minimum time interval between successive initiation events (i.e. the "eclipse").;The gidA and mioC genes, which flank the chromosomal origin of replication, and the dnaA gene, which encodes the primary initiator protein, were found to be transcribed in a cell cycle-dependent manner which correlated with the time of initiation of chromosome replication. Transcription of the gidA and dnaA genes were found to reach maxima at the time of initiation, consistent with their proposed positive roles in initiation, and to be inhibited immediately following initiation. Transcription through oriC emanating from the mioC gene was found to be inhibited prior to initiation due to the binding of DnaA protein within its promoter. This observation was consistent with the idea that mioC transcription may negatively regulate initiation, and therefore could be responsible for preventing initiation during the "eclipse". Further study, however, revealed that replication can initiate with the proper timing even in the presence of continuous mioC transcription. From these results it is proposed that the inhibition of mioC transcription serves as an index of the minimum time needed to establish active DnaA protein/DNA complexes within oriC.;The results of this study also demonstrate that the transient inhibitions of gidA and dnaA transcription following initiation are due to sequestration of their promoters at the cell membrane. Conversely, the mioC promoter was found to be sequestered for a very short time or not all, thus revealing that mioC transcription traverses oriC while oriC is still sequestered at the cell membrane following initiation. Furthermore, the absence of sequestration of the oriC region was found to reduce the duration of the eclipse period, indicating that sequestration functions to prevent immediate reinitiation. Like oriC, NR1 plasmids and the ColE1 plasmid pBR322 were also shown to experience an eclipse period following their replication. However, unlike oriC, these plasmids replicated throughout the cell division cycle. The data presented in this study were used to develop a model which further defines the series of molecular events involved in regulating the initiation of DNA replication during the E. coli division cycle.