Replication Control in Plasmid R1: Duplex Formation Between the Antisense RNA, CopA, and Its Target, CopT, is Not Required for Inhibition of RepA Synthesis

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 1992 Mar 1

PMID 1372249

Citations 20

Authors

E G WAGNER

P Blomberg

K Nordstrom

Affiliations

Soon will be listed here.

Abstract

The replication frequency of plasmid R1 is regulated by an antisense RNA, CopA, which inhibits the synthesis of the rate-limiting initiator protein RepA. The inhibition requires an interaction between the antisense RNA and its target, CopT, in the leader of the RepA mRNA. This binding reaction has previously been studied in vitro, and the formation of a complete RNA duplex between the two RNAs has been demonstrated in vitro and in vivo. Here we investigate whether complete duplex formation is required for CopA-mediated inhibition in vivo. A mutated copA gene was constructed, encoding a truncated CopA which is impaired in its ability to form a complete CopA/CopT duplex, but which forms a primary binding intermediate (the 'kissing complex'). The mutated CopA species (S-CopA) mediated incompatibility against wild-type R1 plasmids and inhibited RepA-LacZ fusion protein synthesis. Northern blot, primer extension and S1 analyses indicated that S-CopA did not form a complete duplex with CopT in vivo since bands corresponding to RNase III cleavage products were missing. An in vitro analysis supported the same conclusion. These data suggest that formation of the 'kissing complex' suffices to inhibit RepA synthesis, and that complete CopA/CopT duplex formation is not required. The implications of these findings are discussed.

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References

Krinke L, WULFF D . The cleavage specificity of RNase III. Nucleic Acids Res. 1990; 18(16):4809-15. PMC: 331951. DOI: 10.1093/nar/18.16.4809. View

Simons R, Kleckner N . Biological regulation by antisense RNA in prokaryotes. Annu Rev Genet. 1988; 22:567-600. DOI: 10.1146/annurev.ge.22.120188.003031. View

Andersen J, Delihas N . micF RNA binds to the 5' end of ompF mRNA and to a protein from Escherichia coli. Biochemistry. 1990; 29(39):9249-56. DOI: 10.1021/bi00491a020. View

Berzal-Herranz A, WAGNER E, Diaz-Orejas R . Control of replication of plasmid R1: the intergenic region between copA and repA modulates the level of expression of repA. Mol Microbiol. 1991; 5(1):97-108. View

Krinke L, WULFF D . RNase III-dependent hydrolysis of lambda cII-O gene mRNA mediated by lambda OOP antisense RNA. Genes Dev. 1990; 4(12A):2223-33. DOI: 10.1101/gad.4.12a.2223. View

Masukata H, Tomizawa J . Control of primer formation for ColE1 plasmid replication: conformational change of the primer transcript. Cell. 1986; 44(1):125-36. DOI: 10.1016/0092-8674(86)90491-5. View

Dong X, Womble D, Rownd R . Transcriptional pausing in a region important for plasmid NR1 replication control. J Bacteriol. 1987; 169(12):5353-63. PMC: 213958. DOI: 10.1128/jb.169.12.5353-5363.1987. View

Polisky B . ColE1 replication control circuitry: sense from antisense. Cell. 1988; 55(6):929-32. DOI: 10.1016/0092-8674(88)90235-8. View

Novick R, Iordanescu S, Projan S, Kornblum J, Edelman I . pT181 plasmid replication is regulated by a countertranscript-driven transcriptional attenuator. Cell. 1989; 59(2):395-404. DOI: 10.1016/0092-8674(89)90300-0. View

10.

TAMM J, Polisky B . Characterization of the ColE1 primer-RNA1 complex: analysis of a domain of ColE1 RNA1 necessary for its interaction with primer RNA. Proc Natl Acad Sci U S A. 1985; 82(8):2257-61. PMC: 397536. DOI: 10.1073/pnas.82.8.2257. View

11.

Taylor J, Schmidt W, Cosstick R, Okruszek A, Eckstein F . The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. Nucleic Acids Res. 1985; 13(24):8749-64. PMC: 318949. DOI: 10.1093/nar/13.24.8749. View

12.

Taylor J, Ott J, Eckstein F . The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985; 13(24):8765-85. PMC: 318950. DOI: 10.1093/nar/13.24.8765. View

13.

Melton D, Krieg P, Rebagliati M, Maniatis T, Zinn K, Green M . Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984; 12(18):7035-56. PMC: 320141. DOI: 10.1093/nar/12.18.7035. View

14.

Maxam A, Gilbert W . Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980; 65(1):499-560. DOI: 10.1016/s0076-6879(80)65059-9. View

15.

Nordstrom K, Molin S . Partitioning of plasmid R1 in Escherichia coli. II. Incompatibility properties of the partitioning system. Plasmid. 1980; 4(3):332-9. DOI: 10.1016/0147-619x(80)90071-2. View

16.

Stougaard P, Light J, Molin S . Convergent transcription interferes with expression of the copy number control gene, copA, from plasmid R1. EMBO J. 1982; 1(3):323-8. PMC: 553043. DOI: 10.1002/j.1460-2075.1982.tb01168.x. View

17.

Molin S, Stougaard P, Uhlin B, Gustafsson P, Nordstrom K . Clustering of genes involved in replication, copy number control, incompatibility, and stable maintenance of the resistance plasmid R1drd-19. J Bacteriol. 1979; 138(1):70-9. PMC: 218239. DOI: 10.1128/jb.138.1.70-79.1979. View

18.

Ma C, Simons R . The IS10 antisense RNA blocks ribosome binding at the transposase translation initiation site. EMBO J. 1990; 9(4):1267-74. PMC: 551804. DOI: 10.1002/j.1460-2075.1990.tb08235.x. View

19.

Blomberg P, WAGNER E, Nordstrom K . 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. EMBO J. 1990; 9(7):2331-40. PMC: 551961. DOI: 10.1002/j.1460-2075.1990.tb07405.x. View

20.

Persson C, WAGNER E, Nordstrom K . Control of replication of plasmid R1: formation of an initial transient complex is rate-limiting for antisense RNA--target RNA pairing. EMBO J. 1990; 9(11):3777-85. PMC: 552136. DOI: 10.1002/j.1460-2075.1990.tb07591.x. View