Probing the sequence and structure of in vitro synthesized antisense and target RNAs from the replication control system of plasmid pMV158

•Antisense and target RNAs of the pMV158 replication control were produced in vitro.•A full-length antisense RNA was the main run-off transcription product.•Longer-than-expected target RNAs were the main run-off transcription products.•Folding models were established for the antisense and target RNAs.•The antisense RNA bound efficiently to the two target transcripts.

Antisense RNAII is a replication control element encoded by promiscuous plasmid pMV158. RNAII binds to its complementary sequence in the copG-repB mRNA, thus inhibiting translation of the replication initiator repB gene. In order to initiate the biochemical characterization of the pMV158 antisense RNA-mediated control system, conditions for in vitro transcription by T7RNA polymerase were set up that yielded large amounts of antisense and target run-off products able to bind to each other. The run-off antisense transcript was expected, and confirmed, to span the entire RNAII as synthesized by the bacterial RNA polymerase, including the intrinsic transcription terminator at its 3′-terminus. On the other hand, two different target transcripts, mRNA60 and mRNA80, were produced, characterized and tested for efficient binding to the antisense product. The mRNA60 and mRNA80 run-off transcripts supposedly spanned 60 and 80 nucleotides, respectively, on the copG-repB mRNA and lacked terminator-like structures at their 3′-termini. Probing of the sequence and conformation of the main products, along with modeling of their secondary structures, showed that both target transcripts were actually longer-than-expected, and contained a 3′-terminal hairpin wherein the extra nucleotides base-paired to the expected 3′-terminus of the corresponding run-off transcript. These longer products were proposed to arise from the RNA-dependent polymerizing activity of T7RNA polymerase on correct run-off transcripts primed by extremely short 3′-selfcomplementarity. Seizing of the target mRNA sequence complementary to the 5′-terminus of RNAII in a stable 3′-terminal hairpin generated by this activity seemed to cause a 3-fold decrease in the efficiency of binding to the antisense RNA.