Autoregulation-deficient mutant of the plasmid R6K-encoded pi protein distinguishes between palindromic and nonpalindromic binding sites.

D York,M Filutowicz

doi:10.1016/s0021-9258(20)80619-x

Abstract

The autogenously regulated gene pir of Escherichia coli plasmid R6K encodes the replication protein pi. This protein binds to two sites in the operator region of the pir gene: a 22-base pair nonpalindromic sequence and a pair of palindromic 9-base pair sequences. These pi-binding sites are similar, suggesting that pi uses a single DNA-binding domain in recognizing them. We devised a plasmid system permitting isolation of mutants of the pi protein which are altered in autoregulation. A Ser87 to Asn substitution in one such mutant, designated pi 87, reduces the protein's ability to repress the pir gene promoter in vivo. DNase I protection and gel retardation assays were carried out with highly purified pi 87 protein. In these studies pi 87 exhibited altered binding to the palindromic but not to the nonpalindromic part of the operator of the pir gene. Chemical cross-linking and gel filtration analyses have shown that the dimerization properties of wild type pi and pi 87 proteins are similar in solution. We propose that the interaction of pi protein with the palindromic part of the pir operator is essential for autoregulation; we also propose that there is a fundamental difference in the mechanisms of pi protein recognition of palindromic and nonpalindromic sequences.

Highlights

The autogenously regulated gene pir of Escherichia operator of thepir gene (Germino and Bastia,1983;Filutowicz coli plasmid R6K encodes the replication protein u. et al, 1985~)
The Gs at the second and fourth positions of this motif are critical for K binding to repeats,as shown by mutational analysis (McEachern et al, 1985) and dimethyl sulfate methylation protection experiments carried out both in vivo2 and in vitro (Germino and Bastia, 1983).These data strongly suggest that a single DNA binding domain of K
Exhibited altered binding to the palindromicbut not to K protein autogenously regulates its own synthesis at the the nonpalindromicpart of the operator of thepir gene. transcriptional level (Filutowicz et al, 1985a;Kelley and Chemicalcross-linking and gel filtration analyses haveBastia, 1985).It is not clear which of the two P binding sites shown that the dimerization properties of wild type u in the pir gene operator is essential for autoregulation

Summary

Introduction

The autogenously regulated gene pir of Escherichia operator of thepir gene (Germino and Bastia,1983;Filutowicz coli plasmid R6K encodes the replication protein u. et al, 1985~). Transcriptional level (Filutowicz et al, 1985a;Kelley and Chemicalcross-linking and gel filtration analyses haveBastia, 1985).It is not clear which of the two P binding sites shown that the dimerization properties of wild type u in the pir gene operator is essential for autoregulation.

Results

Conclusion