Antisense RNA Control of Plasmid R1 Replication

The replication frequency of plasmid R1 is controlled by an antisense RNA (CopA) that binds to its target site (CopT) in the leader region of repA mRNA and inhibits the synthesis of the replication initiator protein RepA. Previous studies on CopA-CopT pairing in vitro revealed the existence of a primary loop-loop interaction (kissing complex) that is subsequently converted to an almost irreversible duplex. However, the structure of more stable binding intermediates that lead to the formation of a complete duplex was speculative. Here, we investigated the interaction between CopA and CopT by using Pb(II)-induced cleavages. The kissing complex was studied using a truncated antisense RNA (CopI) that is unable to form a full duplex with CopT. Furthermore, RNase III, which is known to process the CopA-CopT complex in vivo, was used to detect the existence of a full duplex. Our data indicate that the formation of a full CopA-CopT duplex appears to be a very slow process in vitro. Unexpectedly, we found that the loop-loop interaction persists in the predominant CopA-CopT complex and is stabilized by intermolecular base pairing involving the 5′-proximal 30 nucleotides of CopA and the complementary region of CopT. This almost irreversible complex suffices to inhibit ribosome binding at the tap ribosome binding site and may be the inhibitory complex in vivo.

[1]  J. Tomizawa.  Control of cole 1 plasmid replication: The process of binding of RNA I to the primer transcript , 1984, Cell.

[2]  S. Molin,et al.  Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the plasmid R1 system. , 1984, Plasmid.

[3]  E. Wagner,et al.  The effect of loop size in antisense and target RNAs on the efficiency of antisense RNA control. , 1992, Nucleic acids research.

[4]  Secondary structure analysis of the RepA mRNA leader transcript involved in control of replication of plasmid R1. , 1989, Nucleic acids research.

[5]  A. Nicholson,et al.  Defining the enzyme binding domain of a ribonuclease III processing signal. Ethylation interference and hydroxyl radical footprinting using catalytically inactive RNase III mutants. , 1996, The EMBO journal.

[6]  E. Wagner,et al.  Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation. , 1992, The EMBO journal.

[7]  J. Tomizawa.  Control of ColE1 plasmid replication. Intermediates in the binding of RNA I and RNA II. , 1990, Journal of molecular biology.

[8]  E. Wagner,et al.  Control of replication of plasmid R1: structures and sequences of the antisense RNA, CopA, required for its binding to the target RNA, CopT. , 1990, The EMBO journal.

[9]  Stanley N Cohen,et al.  The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo , 1991, Cell.

[10]  E. Wagner,et al.  Replication control of plasmid R1: disruption of an inhibitory RNA structure that sequesters the repA ribosome‐binding site permits tap‐independent RepA synthesis , 1994, Molecular microbiology.

[11]  E. Wagner,et al.  Control of replication of plasmid R1: kinetics of in vitro interaction between the antisense RNA, CopA, and its target, CopT. , 1988, The EMBO journal.

[12]  R. Simons,et al.  Antisense RNA control in bacteria, phages, and plasmids. , 1994, Annual review of microbiology.

[13]  E. Wagner,et al.  Control of replication of plasmid R1: formation of an initial transient complex is rate‐limiting for antisense RNA–target RNA pairing. , 1990, The EMBO journal.

[14]  E. Wagner,et al.  Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III. , 1990, The EMBO journal.

[15]  D. Crothers,et al.  Determinants of RNA hairpin loop-loop complex stability. , 1995, Journal of molecular biology.

[16]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[17]  A. Pittard,et al.  Mutations affecting pseudoknot control of the replication of B group plasmids , 1993, Journal of bacteriology.

[18]  E. Wagner,et al.  An antisense/target RNA duplex or a strong intramolecular RNA structure 5' of a translation initiation signal blocks ribosome binding: the case of plasmid R1. , 1996, RNA.

[19]  E. Wagner,et al.  Bulged-out nucleotides in an antisense RNA are required for rapid target RNA binding in vitro and inhibition in vivo. , 1995, Nucleic acids research.

[20]  E. Wagner,et al.  Antisense RNA‐mediated transcriptional attenuation occurs faster than stable antisense/target RNA pairing: an in vitro study of plasmid pIP501. , 1994, The EMBO journal.

[21]  J. Tomizawa.  Control of ColE1 plasmid replication: Binding of RNA I to RNA II and inhibition of primer formation , 1986, Cell.

[22]  K. Siemering,et al.  Mechanism of binding of the antisense and target RNAs involved in the regulation of IncB plasmid replication , 1994, Journal of bacteriology.

[23]  E. Wagner,et al.  Kinetic aspects of control of plasmid replication by antisense RNA. , 1994, Trends in biochemical sciences.

[24]  E. Wagner,et al.  Bulged-out nucleotides protect an antisense RNA from RNase III cleavage. , 1995, Nucleic acids research.

[25]  D. Crothers,et al.  Bent helix formation between RNA hairpins with complementary loops. , 1995, Science.

[26]  E G Wagner,et al.  Replication control in plasmid R1: duplex formation between the antisense RNA, CopA, and its target, CopT, is not required for inhibition of RepA synthesis. , 1992, The EMBO journal.