## 4 Discussion  
Significant effects of the _E. coli_ Hfq protein on the regulation of ColE1-type plasmid DNA replication have been proposed based on genetic studies [17,18]. Namely, the efficiency of transformation of _E. coli hfq_ mutant cells with a ColE1-like plasmid was decreased significantly relative to isogenic wild-type cells [17,18]. Such results suggest a positive regulation of plasmid DNA replication by Hfq. However, a higher efficiency of plasmid DNA synthesis occurred in the absence of the active Hfq protein in host cells [17], which is rather compatible with an opposite effect, i.e., inhibition of ColE1 DNA replication by Hfq. Recent studies on _E. coli_ transformation by ColE1-like plasmids suggested that Hfq might act at the stage of establishment of plasmid DNA replication after the plasmid has entered the cell [18]. Clearly, the effects of Hfq on the regulation of ColE1 plasmid replication are complex and possibly involve different stages and reactions which are not yet determined. Figure 11: ColE1 C-stretch temperature melting using SRCD (from blue 15 \({}^{\circ}\)C to red 95 \({}^{\circ}\)C). (**A**) Melting with the CTR. (**B**) Melting without the CTR. At 15 \({}^{\circ}\)C, ColE1 C-stretch forms a B helix: the correlation between spectra at 15 \({}^{\circ}\)C and spectra of the B-form helix was investigated using Pearson correlation tests. We found a correlation of 0.9052 for a \(p\)-value of 1.29063 \(\times\) 10\({}^{-57}\) for colE1 C-stretch and a correlation of 0.95848 for a \(p\)-value of 1.23619 \(\times\) 10\({}^{-83}\) for colE1 C-stretch in the presence of the CTR. (**C**) The corresponding T\({}_{\mathrm{m}}\) were 81.4 \(\pm\) 0.3 \({}^{\circ}\)C, 73.4 \(\pm\) 0.3 \({}^{\circ}\)C, 85.4 + 5 \(\pm\) 1 \({}^{\circ}\)C and 73.4 \(\pm\) 1 \({}^{\circ}\)C at 188 (blue) and 275 (red) nm with (full circle) and without the CTR (empty circle), respectively. In light of the above-described uncertainty regarding the specific role(s) of Hfq in ColE1-type plasmid replication and considering the recently identified effects of CTR of this protein on plasmid biology [18], it is clear that detailed studies on interactions of different domains of Hfq with NA involved in ColE1 DNA replication are crucial. Such studies are also important because plasmid-borne antibiotic resistance is a serious medical problem; thus, understanding details of molecular mechanisms of plasmid DNA replication regulation may indicate ways to limit this very adverse phenomenon [3; 4]. On the other hand, methods for precise studies on interactions between small proteins and NA are restricted. Results of experiments are often difficult to interpret due to the limited resolutions of available laboratory techniques. Therefore, in this work, we have developed SRCD-based methods of investigation of interactions of Hfq, and especially its CTR, with NA. This allowed us to assess the effects of the tested protein on interactions between oligonucleotides representing crucial fragments of NA involved in the regulation of ColE1 plasmid replication. SRCD was employed in these studies, as the synchrotron light source could provide intensities of the beam below 190 nm, allowing the determination of NA conformation. The crucial results of our studies on the interactions of CTR of Hfq with NA involved in the regulation of ColE1 plasmid DNA replication can be summarized as follows. First, CTR does not melt dsDNA at the ColE1 origin; conversely, it significantly stabilizes the dsDNA ColE1 origin structure. Second, CTR aligns the ColE1 origin dsDNA. Third, CTR accelerates RNA I-RNA II annealing. Fourth, CTR aligns the RNA I-RNA II duplex. Fifth, CTR stabilizes the RNA I-RNA II duplex, especially at high temperatures. Sixth, CTR stabilizes RNA II secondary structure but not that of RNA I. Seventh, CTR does not melt the C-stretch region of ColE1 plasmid DNA; conversely, it stabilizes it.