Initiation of plasmid R1 replicationin vitrois independent of transcription by host RNA polymerase | Zendy

Ramón Díaz | Zendy; Sagrario Ortega | Zendy

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Initiation of plasmid R1 replicationin vitrois independent of transcription by host RNA polymerase

Author(s) -

Ramón Díaz,

Sagrario Ortega

Publication year - 1984

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/12.13.5175

Subject(s) - biology , transcription (linguistics) , rna polymerase , polymerase , rna polymerase ii , t7 rna polymerase , microbiology and biotechnology , plasmid , genetics , rna , gene , promoter , gene expression , philosophy , linguistics , escherichia coli , bacteriophage

A Sau3A fragment containing most of the repA gene of plasmid R1 has been cloned in the BamH1 site of the expression vector pAS1. One of the recombinants, pSO1, codes for a fused RepA' protein which is efficiently synthesized both in vivo and in vitro from transcriptional and translational signals of the vector. It is shown that following pSO1 promoted accumulation of RepA' in cell-free extracts of E. coli, in vitro replication of the R1 miniplasmid pKN182 can initiate and proceed uncoupled from further protein synthesis. Using this uncoupled system and also a M13mp9 based ori-R1 recombinant, pRD95, it is also shown that RepA' acts at the origin region of R1 to promote initiation of replication that is independent on transcription by host RNA polymerase. This is indicated by the insensitivity of pRD95 and pKN182 replication to rifampicin as well as to RNA polymerase antibodies. The properties of the uncoupled in vitro replication system are further described.

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