| Abstract [eng] |
The aim of this study was the investigation of interaction of wild type restriction endonuclease (REase) Eco31I and mutant REase Eco31IQ69R/K127I with DNA sequences 5’-GGTCTC-3’, 5’-AGTCTC-3’, 5’-TGTCTC-3’ and 5’-CGTCTC-3 by an in vivo transcriptional interfererence assay. The transcriptional interference assay is composed of an aadA gene that determines the resistance to spectinomycin and antisense constitutive E. coli promoter conII. The expression of aadA gene is blocked by the transcription from promoter conII and, therefore, the phenotype is SpS. The promoter conII could be regulated by installing operator sequence. DNA-binding protein binds to this recognition sequence and acts as a repressor of promoter conII. This interaction determines transformation of the phenotype to SpR. Four transcriptional interference system plasmids with operon sequences 5’-GGTCTC-3’, 5’-AGTCTC-3’, 5’-TGTCTC-3’and 5’-CGTCTC-3’ were engineered during this study. Cleavage deficient variants Eco31IH312A and Eco31IQ69R/K127I+H312A were tested, because the presence of REase in the absence of cognate DNA methyltransferase is lethal to the host. We predicted that the both enzymes (Eco31IH312A and Eco31IQ69R/K127I+H312A) would interact with 5’-GGTCTC-3’ sequence. Therefore, the interaction of Eco31IQ69R/K127I+H312A with sequences 5’-AGTCTC-3’ and 5’-TGTCTC-3’ at the lower level was expected. The optimal conditions of transcriptional interference were selected and the both enzymes interacted only with 5’-GGTCTC-3’ sequence. The expected interaction of Eco31IQ69R/K127I+H312A with 5’-AGTCTC-3’ or 5’-TGTCTC-3’ sequences was insufficient to prevent transcription from promoter conII. |
