Co-operative autoregulation of a replication protein gene

In this work we present the localization and characterization of the repl promoter (Prepl) and show aspects of the regulation. Comparison of Prepl with other autoregulated replication protein gene promoters revealed similarities, but Prepl differs from some of these characterized promoters in not being regulated by the heat-shock RNA polymerase. Primer extension analysis showed that Prepl is contained within five helically aligned 18 base pair repeats, or 18-mers of the previously defined minimal origin. In addition, we find that Prepl is autoregulated by a trans-acting product encoded in the REPI region. Purified Repl protein binds to the 18-mer region of the origin, suggesting that the repl gene is autoregulated by the protein product. The autoregulation appears to be co-operative since decreasing the 18-mer binding site region results in a concomitant non-linear loss of autorepression. The deletion derivatives show a decreased ability to bind the Repl protein when compared with origin DNA containing all of the binding region. The diminished capacity of the various deletion derivatives to bind Repl in vitro correlates with the loss of autorepression seen in vivo.