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

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RESUMO

Plasmids 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 antisense 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 inactivation of RepU. Polypeptides with apparent molecular masses of 42 (RepU*) and 39 (RepU) kDa were purified, both having the N-terminal 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 RepU 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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