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Fig. 1. The ColE1 origin. (a) X-ray crystal structure of Rop (Banner et al., 1987) rendered in MOLSCRIPT (Kraulis, 1991) from 1ROP. (b) Schematic representation of the interaction between RNA I (108 nt) and RNA II (550 nt after processing) (Cesareni et al., 1991). (c) Examples of small stem–loop RNAs derived from the complex in (b), used in gel-shift assays for Rop function (Predki et al., 1995). (d) Organization of the ColE1 origin. RNA I and RNA II are divergently transcribed, allowing the inhibitor RNA to bind in a complementary fashion to the priming RNA. The position of the G/A polymorphism between pBR322 and pUC19 is noted.
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Fig. 2. Vector maps. (a) The pAClacRop plasmid expresses Rop from a synthetic lac promoter. The p15A origin and kanamycin resistance marker make it compatible with the pUC...GFPuv vectors. Rop is replaced with a short linker in pAClacLink and the gene for CAT in pAClacCm. (b) The pUCBADGFPuv plasmid expresses GFPuv (Crameri et al., 1996) from the arabinose promoter. It bears the pUC19 version of the ColE1 origin and the ampicillin resistance marker. In pUClacGFPuv, the arabinose promoter and araC gene are replaced by the lac promoter from pUC19.
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Fig. 3. Screening for Rop function. (a) The principle of the negative screen is that active Rop lowers the copy number of the ColE1 plasmid from which GFP is expressed, reducing cellular fluorescence. (b) Negative screen. DH10B E.coli transformed with pUClacGFPuv and pAClacRop (left) or pAClacLink (right) were grown for 16 h at 42°C on LB agar supplemented with kanamycin and ampicillin. Cells are visualized under long-wave UV light. (c) Positive screen. DH10B E.coli transformed with pUCBADGFPuv and pAClacRop (left) or pAClacLink (right) were grown for 16 h at 42°C on LB agar supplemented with 0.0005% arabinose, 100 µM IPTG, kanamycin and ampicillin. Cells are visualized under long-wave UV light. (d) Plasmid minipreps from the positive screen. A 1% agarose gel of XmaI/AatII-cleaved DNA minipreps from 200 µl of liquid culture grown in LB supplemented as with the plates above. Lane 1, 
BstEII marker (NEB); lane 2, pAClacRop/pUCBADGFPuv DH10B (bright cells); lane 3, pAClacLink/pUCBADGFPuv DH10B (dim cells). The double scission of the smaller plasmid in lane 3 confirms that it is pAClacLink.
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Fig. 4. Systematically repacked Rop variants. (a) Schematic representation of the five repacked Rop variants previously designed by Munson et al. (1994a, 1996). Large closed circles represent Leu, small closed circles represent Ala, and closed ovals represent Met. The nomenclature represents the composition of each layer of the core (e.g. Ala2Leu2), the number of layers that are repacked (i.e. 2, 4 or 8), and whether or not the residue size alternation is maintained or reversed (i.e. rev) in the penultimate layers. (b) Positive screen (growth and visualization as in Figure 3) of the repacked Rop variants. Owing to the position of the UV lamp in the lower-left corner of the plate (necessary for photography), the Ala2Met2-8 variant appears brighter than it does when the lamp is directly above the plate. (c) DNA minipreps of the same cells cleaved with XmaI. Only Ala2Leu2-2 has strong activity, as evidenced by cellular fluorescence or ColE1 plasmid amount in the miniprep. In contrast, Munson et al. found that all three of these Ala2Leu2-X variants bound model stem–loop RNAs with dissociation constants comparable with wt Rop.
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Fig. 5. Poor NdeI scission of pAClac and gel separation of pAClacCm/NdeI/BanI scission products. (a) Scission of 50 ng of pAClacLink plasmid for 1 h at 37°C with NdeI. Lane 1, BstEII marker; lanes 2–6, scission with 0, 5, 10, 20 and 40 U of NdeI. The ratio of units of enzyme to amount of DNA is shown below each lane (typically, 1x is sufficient under these conditions). The plasmid preparation, which contains slow-running nicked DNA and fast-running supercoiled DNA, can be seen to resolve to a single, intermediately running linearized species after 400–800-fold scission. A normal amount of BanI is sufficient for complete linearization (data not shown). (b) Agarose gel of NdeI/BanI digested pAClacCm plasmid. Lane 1, BstEII marker; lane 2, singly (slow) and doubly cut (fast) material can easily be resolved on a 1% agarose gel; lane 3, a portion of a preparative-scale lane from which the doubly cut material was removed by electrophoresis onto a dialysis membrane.
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