C.EcoO1091, a regulatory protein for production of EcoO1091 restriction endonuclease, specfically binds to and bends DNA upstream of its translational start site

Abstract

The EcoO109l restriction-modification system, which recognizes 5′-(A/G)GGNCC(C/T)-3′, has been cloned, and contains convergently transcribed endonuclease and methylase. The role and action mechanism of the gene product, C.EcoO109l, of a small open reading frame located upstream of ecoO109lR were investigated in vivo and in vitro. The results of deletion analysis suggested that C.EcoO109l acts as a positive regulator of ecoO109lR expression but has little effect on ecoO109lM expression. Assaying of promoter activity showed that the expression of ecoO109lC was regulated by its own gene product, C.EcoO109l. C.EcoO109l was overproduced as a Histag fusion protein in recombinant Escherichia coli HB101 and purified to homogeneity. C.EcoO109l exists as a homodimer, and recognizes and binds to the DNA sequence 5′-CTAAG(N)5CTTAG-3′ upstream of the ecoO109lC translational start site. It was also shown that C.EcoO109l bent the target DNA by 54 ± 4°.