C.EcoO109I, a regulatory protein for production of EcoO109I restriction endonuclease, specifically binds to and bends DNA upstream of its translational start site

Abstract

The EcoO109I 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.EcoO109I, of a small open reading frame located upstream of ecoO109IR were investigated in vivo and in vitro. The results of deletion analysis suggested that C.EcoO109I acts as a positive regulator of ecoO109IR expression but has little effect on ecoO109IM expression. Assaying of promoter activity showed that the expression of ecoO109IC was regulated by its own gene product, C.EcoO109I. C.EcoO109I was overproduced as a His-tag fusion protein in recombinant Escherichia coli HB101 and purified to homogeneity. C.EcoO109I exists as a homodimer, and recognizes and binds to the DNA sequence 5'-CTAAG(N)(5)CTTAG-3' upstream of the ecoO109IC translational start site. It was also shown that C.EcoO109I bent the target DNA by 54 +/- 4 degrees.