C.EcoO109I, a regulatory protein for production of EcoO109I restriction endonuclease, specifically binds to and bends DNA upstream of its translational start site.
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.
Kita
Keiko
K
Department of Biotechnology, Tottori University, 4-101 Koyama, Tottori 680-8552, Japan. kita@bio.tottori-u.ac.jp
Tsuda
Junko
J
Nakai
Shin-ya
SY
D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics*; D020218:Response Elements / Q000235:genetics; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D015252:Deoxyribonucleases, Type II Site-Specific; C045450:RGGNCCY-specific type II deoxyribonucleases