## 4 Discussion
How can the above-summarized results, and demonstrated properties of CTR, be interpreted in the light of in vivo results which might lead to ostensibly contradictory conclusions about the positive vs. negative regulation of ColE1 replication by Hfq? In fact, CTR revealed properties that might both potentially stimulate DNA replication by aligning the dsDNA origin region (thus making the DNA region more susceptible to further specific interactions with pre-primer RNA and subsequent replication initiation) and inhibit the initiation of this process by the stimulation and stabilization of RNA I-RNA II interactions. On the other hand, no effects clearly suggesting activation of the replication process by CTR could be observed, e.g., melting of the C-stretch DNA region or annealing of RNA II to this region. In fact, CTR even stabilized the dsDNA duplex in two regions of the ColE1 origin, which suggests an inhibitory effect on DNA replication initiation. Therefore, one might propose that Hfq, or more precisely, its CTR, is probably a negative rather than positive regulator of ColE1-type plasmid DNA replication. Such a proposal is compatible with the finding that in the _Hfq_ mutant, the efficiency of synthesis of ColE1-like plasmid DNA was higher than in the wild-type control cells [17]. However, a decreased efficiency of transformation of mutants devoid of either the whole _Hfq_ gene or its part coding for CTR by ColE1-like plasmids [18] might suggest a seemingly opposite conclusion, i.e., that Hfq positively affects ColE1 plasmid replication. Nevertheless, one should note that such a suggestion is based on the most frequently accepted interpretation of results of the efficiency of transformation measurement, assuming that a lower number of host cells can be transformed if plasmid replication is impaired. However, it was demonstrated previously that if negative control of plasmid replication is abolished, the plasmid DNA synthesis process proceeds extremely efficiently. This so-called runaway replication causes non-controlled overproduction of plasmid DNA in cells leading to adverse effects, including cell death [51]. Such a phenomenon was also observed in _E. coli_ cells bearing ColE1-type plasmids, though the final effect was a decreased viability of bacteria, from about 2-fold [52] to about 10-fold [53], rather than the complete elimination of living cells. Therefore, we suggest that less efficient negative control of plasmid replication initiation by Hfq, observed in _E. coli__Hfq_ mutants [17; 18], might result in runaway replication of ColE1-like plasmid and impaired viability of transformed bacteria. If this hypothesis is true, the decreased number of _hfq_ mutant transformants appearing in experiments with these plasmids might be compatible with the role of Hfq in the negative regulation of plasmid replication initiation, as the over-replication (confirmed by the higher efficiency of incorporation of labeled precursors (nucleotides) into plasmid DNA) would cause lower viability of bacteria that acquired plasmids. An independent, intriguing observation demonstrated in this study was that CTR of Hfq efficiently stabilized RNA I-RNA II interactions under extreme temperature conditions (above 80 \({}^{\circ}\)C). This effect might be related to the thermostability of Hfq. However, the annealing of oligonucleotides is highly sensitive to temperature; thus, such a phenomenon is surprising. Obviously, 80 \({}^{\circ}\)C cannot be applied to in vivo conditions, as _E. coli_ dies at temperatures over 54 \({}^{\circ}\)C [54]. Nevertheless, in _E. coli_ cells, effects of Hfq could be more pronounced at temperatures like 42-45 \({}^{\circ}\)C, while at very high temperatures, we observed enhanced effects which cannot occur in living cells, but in vitro, they were clearly represented. One might speculate that the stabilization effect at the high temperature arises from the formation of high-molecular-weight, amyloid-like complexes, formed by Hfq and its CTR.