# 60 Years of Studies into the Initiation of Chromosome Replication in Bacteria
## 5 \"The Initiation Mess\" Revisited: Conundrums
### The In Vitro/In Vivo RNA Polymerase Conundrum  
The absolute dependence on RNA polymerase during in vivo cell growth is the salient feature distinguishing the in vitro from the in vivo replication initiation system. It has long been assumed that RNA polymerase is essential because the in vivo system might depend on RNA polymerase to prime initiation for DNA synthesis at _oriC_, in the manner that Okazaki fragments require RNA primer to initiate lagging strand DNA synthesis. Evidence for an active role of RNA polymerase in the initiation of DNA replication resides principally in the observation that inhibiting RNA synthesis with rifampicin blocks DNA synthesis [102]. As mentioned above, no such primer has been identified as being essential for replication initiation at _oriC_[103]. Transcription from genes flanking _oriC_--_mioC_ and _gidA_--modulates replication initiation by introducing positive or negative DNA supercoiling. However, the products of these genes themselves are not essential, and the transcription of these regions is not required for initiation. In contrast, a mutation in _rpoB_ suppresses the _dnaA46_ temperature-sensitive mutation, while a mutation in _rpoC_ results in a two-fold-to-three-fold increase in DNA concentration in a replicon-dependent manner [104]. The behaviour of these two mutants is consistent with a direct interaction between RNA polymerase and DnaA at _oriC_, an interaction that would be essential in vivo but dispensable in vitro. However, the increase in the chromosome number might be explained by an overall effect on cellular transcription rather than RNA polymerase acting specifically at or near _oriC_[105]. Progression through the _E. coli_ cell cycle requires de novo protein synthesis and de novo DnaA synthesis for each division cycle. De novo protein synthesis might explain the need for RNA polymerase in vivo but not in vitro, where the requisite biochemical components are in abundant supply. The in vivo dependence on de novo protein synthesis would seem to imply that essential metabolites are exhausted during cell growth, certain proteins and enzymes need to be recycled for key structures to be re-assembled (and/or disassembled), or that the process itself of translation is required (see below). Interestingly, the increase in the chromosomal copy number in the _rpoC_ mutant corresponds to a five-fold increase in DnaA [104], yet hyper-initiation in the mutant does not result in stalled or collapsed forks, in contrast to DnaA-induced hyper-initiation in wild-type and other strains that over-initiate DNA replication [62]. This might suggest that the _rpoC_ mutant allows an over-expression of other genes whose products are required for fork stabilization/restart and/or replication elongation. A possible candidate is Ribonucleotide Reductase (RNR), which supplies dNTPs for DNA synthesis and fork progression (see below). Significantly, the expression of the _urdAB_ genes encoding RNR, for example, is upregulated during DNA replication stress in all organisms examined so far, indicating its importance in DNA repair and replication fork restart [106]. Mutations in _hda_, _dnaA_, and _dnaN_ genes also increase the concentration of RNR [107], presumably by suppressing DnaA inhibition of _urdAB_ gene expression [108]. Mutations in _dnaN_ are known to result in elevated levels of _urdAB_ expression in a strictly Hda-dependent manner [109], consistent with the observation that low DnaA-ATP levels de-repress _urdAB_ expression, while high levels repress transcription and consequently limit/decrease RNR and dNTP pool sizes [110; 111]. Furthermore, transcription rates of _dnaA_ and _urdA_ oscillate during the cell cycle: _urdA_ transcription levels are maximal when _dnaA_ transcription levels are minimal and vice versa (180 degrees out of phase, see Extended Data Figure 6B in [112]), indicating a form of cell size-dependent, regulatory cross-talk between the two genes. The _rpoC_ mutant might, therefore, affect DnaA repression/de-repression of _urdAB_ transcription (see also [109]).