The frequency of replication of IncFII plasmids is regulated by the availability of a rate‐limiting protein, RepA. This protein acts to promote initiation of replication and its synthesis is negatively controlled both at the transcriptional and translational level. The translational control is exerted by the binding of a small antisense RNA, CopA RNA, to its target, CopT, which is located in the leader region of the RepA mRNA. As a consequence, formation of RepA is inhibited. Here we demonstrate the binding of CopA RNA to CopT RNA in vitro; the rate constant of binding was determined to be approximately 1 X 10(6) M‐1 s‐1 at 37 degrees C. We have also shown that in vitro synthesized RepA mRNA molecules differing in length, but which contain the whole CopT region, are able to bind CopA RNA with similar rates. Analysis of the binding of CopA/CopT molecules derived from a copy‐number mutant plasmid showed that the effect of the mutation on the rate of in vitro binding correlates well with its phenotypic effect in vivo, i.e. the binding rate constant is lowered in proportion to the increase in copy number. Likewise, the result of an in vitro incompatibility test is in agreement with in vivo data.

The replication frequency of plasmid R1 is determined by the availability of the RepA protein, which acts at the origin of replication to promote initiation. Synthesis of RepA is negatively regulated both at the transcriptional and post‐transcriptional levels. Post‐transcriptional control is exerted through the action of an antisense RNA, CopA RNA. The target of CopA RNA, CopT RNA, is located in the leader region of the RepA mRNA. Binding between CopA and CopT inhibits repA expression. We have previously presented an in vitro analysis of the binding reaction between CopA and CopT RNAs. In this communication, we extend the in vitro analysis by determining the regions of CopA required for binding, and also demonstrate that binding occurs in at least two steps. The first step is the formation of an initial, transient complex; stem‐loop II is the structure in CopA necessary and sufficient for this step. The subsequent step(s), resulting in the formation of a complete duplex, requires a stretch of single‐stranded nucleotides located 5′ to stem‐loop II in CopA, and its counterpart in CopT. We show that the single‐stranded region can be positioned on either side of stem‐loop II provided that there is a complementary stretch of nucleotides in CopT, indicating that the second step(s) is not sequence‐specific. Furthermore, the effects of salt concentration and temperature on the binding reaction indicate that duplex formation occurs through a mechanism of gradual intra‐strand breaking and inter‐strand formation of hydrogen bonds.

A comparison was made on the use of two spectrophotometric methods, the ferric reducing antioxidant power (FRAP) method and the 2,2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid (ABTS) method, for the measurement of the total antioxidant capacity (TAC) of plant foods. The correlations of TAC measured by the two methods were highly significant in both water-soluble (r2= 0.90) and water-insoluble extracts (r2= 0.98) from 13 strawberry samples. Also a corresponding comparison of TAC in extracts from 14 plant species showed high correlation coefficients, r2= 0.98 for water-soluble extracts and r2= 0.88 for water-insoluble extracts. The ratio of TAC values obtained with the two methods (ABTS/FRAP) varied between 0.7 and 3.3 for different plant extracts indicating that they contained antioxidants with varying reactivity in the two methods. TACs in six pure antioxidant substances were ranked in the following order by both methods: quercetin > ferulic acid > catechin > rutin > caffeic acid > Trolox = chlorogenic acid. The two methods showed similar TAC values for quercetin, rutin, caffeic acid and chlorogenic acid while ferulic acid and catechin gave higher results with the ABTS method than with the FRAP method, and such differences probably explain the varying ratios of ABTS/FRAP obtained in foods. Regarding storage TAC in water-soluble strawberry extracts stored at -20 or -80 degrees C was stable for at least five months while storage at 4 degrees C decreased the TAC value with 40% during five weeks of storage. The study showed that both the ABTS and FRAP methods can be used for convenient monitoring of the antioxidant capacities in fruit and vegetables, and that different antioxidants had varying reactivity in the two methods.

The replication frequency of plasmid R1 is determined by the availability of the initiator protein RepA. Synthesis of RepA is negatively controlled by an antisense RNA, CopA, which forms a duplex with the upstream region of the RepA mRNA, CopT. We have previously shown that the in vitro formation of the CopA‐CopT duplex follows second‐order kinetics and occurs in at least two steps. The first step is the formation of a transient (kissing) complex, which is subsequently converted to a persistent duplex. Here, we investigate the details of the reaction scheme and determine the rate constants of the pathway from the free RNAs to the complete duplex. Using a shortened CopA RNA (CopI) we have been able to determine the association and dissociation rate constants (k1,k‐1) for the kissing complex (which are inferred to be the same for CopI‐T and CopA‐T), and measured the hybridization rate constant k2 (for CopA‐T k2 is at least 1000‐fold greater than for CopI‐T). The analysis of CopA derivatives of mutant and wild‐type origin shows that the rate of formation of the kissing complex is rate‐limiting for the overall pairing reaction between CopA and CopT, both in vitro and in vivo. The biological implications of the kinetically irreversible RNA‐RNA binding reaction scheme are discussed.

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