Abstract
The antisense Inc RNA encoded by the IncIalpha 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 RNA(54), 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 IncIalpha, IncB, and IncFII group plasmids.
Highlights
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
We find that the initial transient interaction (C*) between RepZ mRNA and Inc RNA54, a derivative of Inc RNA lacking its 5Ј-single stranded leader, transforms into a more stable complex altering the sensitivity of the target RNA region to a variety of RNases
Inc RNA54 Binds to Stem-Loop I in Two Steps—We previously synthesized three RNA species, Inc RNA54, Ps14, and SI54 and examined their effects on the interaction between Inc RNA and Rep RNA293, a 293-base RNA corresponding to the repZ mRNA leader (16)
Summary
Incompatibility; RLP, rep leader where Cs is the stable final product, C** is characterized by its altered RNase sensitivity and C* is the unstable complex, the formation of which is inferred from the inhibition kinetics. In the C** complex, base pairing in each loop-loop interaction may stack on the stems of the antisense and target RNAs, which altogether provides the equilibpeptide; RBS, ribosome-binding site. We find that the extensive intermolecluar base pairing is separated at the site of initial base pairing, suggesting a stacking trend between each intermolecular helix and the stem of Inc RNA or its target
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