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Nucleic Acids Research, 1995, Vol. 23, No. 11 1894-1900
© 1995

MOLECULAR BIOLOGY

The level of the pUB110 replication initiator protein is autoregulated, which provides an additional control for plasmid copy number

Anna K. Müller1, Fernando Rojo2 and Juan C. Alonso1,2,*

1Max-Planck-lnstrtut für Molekulare Genetik Ihnestrasse 73, D14195 Berlin, Germany 2Centro Nacional de Biotecnologfa, CSIC, Campus de la Universidad Autbnoma de Madrid Cantoblanco, 28049 Madrid, Spain

* To whom correspondence should be addressed at: Centro Nacional de Biotecnologfa, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received March 1, 1995. Revised April 18, 1995. Accepted April 18, 1995.

Plasmlds control their copy number by limiting the amount of the Initiator for DNA replication. The plasmid pUB110 Initiator protein is termed repU. Expression of the pUB110 repU gene is controlled by two antlsense RNAs that interfere with repU mRNA translation. Genetic evidence suggests that Rep protein levels may be regulated by additional uncharacterized mechanisms. The repU gene product was radiolabeled and purified by monitoring the radioactive label. repU overproduction was performed in cells containing the plasmid leading strand replication origin (dso), to allow for a putative inactlvation of RepU. Polypeptides with apparent molecular masses of 42 (RepU*) and 39 (RepU) kDa were purified, both having the N-termlnal sequence expected for the repU gene. The RepU/repU * protein mixture bound specifically to dso. At low protein concentrations, about six RepU/RepU* protomers bound to the dso region. At higher concentrations, an extended nucleoprotein complex was formed. The promoter for the repU gene was localized downstream of the dso region. The results suggest that the extended RepU/RepU*-dso DNA complex interferes with repU promoter utilization. This provides an additional copy number control by limiting RrepU concentration. Our results suggest that during replication the RepU protein might be converted into an inactive RepU-RepU* hetero-oligomer, further limiting the amount of RepU protein available for replication Initiation.


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