IMPORTANCE OF STRUCTURAL DIFFERENCES BETWEEN COMPLEMENTARY RNA MOLECULES TO CONTROL OF REPLICATION OF AN INCB PLASMID

Replication of the IncB miniplasmid pMU720 is dependent on the express ion of repA, the gene encoding replication initiator protein RepA. Bin ding of a small antisense RNA (RNAI) to its complementary target (stem -loop I [SLI]) in the RepA mRNA prevents the participation of SLI in t he formation of a pseudoknot that is an enhancer of translation of thi s mRNA. Thus, RNAI regulates the frequency of replication of pMU720 by controlling the efficiency of translation of the RepA mRNA. Mutationa l analysis of the two seven-base complementary sequences involved in f ormation of the pseudoknot showed that only the five central bases of each were critical for the formation of the pseudoknot. Physical analy sis of SLI showed that despite the complete complementarity of its seq uence to that of RNAI, the structures of the two molecules are differe nt. The most prominent difference between the two structures is the pr esence of a ii-base internal loop immediately below the hairpin loop o f SLI but not that of RNAI. Closure of this internal loop in SLI resul ted in a 40-fold reduction in repA expression and loss of sensitivity of the residual expression to inhibition by RNAI. By contrast, repA ex pression was largely unaffected by the closure of a lower internal loo p whose presence in SLI and RNAI is essential for effective interactio n between these two molecules. These results suggest that the interact ion of SLI with the distal pseudoknot bases is fundamentally different from the RNAI-SLI binding interaction and that the differences in str ucture between RNAI and SLI are necessary to allow SLI to be able to e fficiently bind RNAI and to participate in pseudoknot formation.