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J Biol Chem, Vol. 275, Issue 2, 1269-1274, January 14, 2000

Structural Analysis of Late Intermediate Complex Formed between Plasmid ColIb-P9 Inc RNA and Its Target RNA
HOW DOES A SINGLE ANTISENSE RNA REPRESS TRANSLATION OF TWO GENES AT DIFFERENT RATES?^*

Katsura Asano^§ and Kiyoshi Mizobuchi^¶

From the  Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan and the ^¶ Department of Applied Physics and Chemistry, University of Electro-Communications, Chofu-shi, Tokyo 182-8585, Japan

The antisense Inc RNA encoded by the IncI ColIb-P9 plasmid replicon controls the translation of repZ encoding the replication initiator and its leader peptide repY at different rates with different mechanisms. The initial loop-loop base pairing between Inc RNA and the target in the repZ mRNA leader inhibits formation of a pseudoknot required for repZ translation. A subsequent base pairing at the 5' leader of Inc RNA blocks repY translation. To delineate the molecular basis for the differential control, we analyzed the intermediate complexes formed between RepZ mRNA and Inc RNA₅₄, a 5'-truncated Inc RNA derivative. We found that the initial base pairing at the loops transforms into a more stable intermediate complex by its propagation in both directions. The resulting extensive base pairing indicates that the inhibition of the pseudoknot formation is established at this stage. Furthermore, the region of extensive base pairing includes bases different in related plasmids showing different incompatibility. Thus, the observed extensive base pairing is important for determining the incompatibility of the low-copy-number plasmids. We discuss the evolution of replication control systems found in IncI, IncB, and IncFII group plasmids.

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