title: Probing the sequence and structure of in vitro synthesized antisense and target RNAs from the replication control system of plasmid pMV158.
abstract: 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.
authors.affiliation: Molecular Microbiology and Infection Biology Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu, 9, 28040 Madrid, Spain.
authors.forename: Celeste
authors.initials: C
authors.lastname: López-Aguilar
authors.forename: Gloria
authors.initials: G
authors.lastname: del Solar
mesh_terms: D016375:Antisense Elements (Genetics) / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D019915:DNA Primase / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D016375:Antisense Elements (Genetics); D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; C074125:RepB' protein, E coli; D019915:DNA Primase; D012319:RNA Polymerase II; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; C121791:dnaG protein, E coli