A BACTERIAL METHYLTRANSFERASE M.ECOHK31I REQUIRES 2 PROTEINS FOR IN-VITRO METHYLATION

The genes encoding EcoHK31l restriction-modification (R-M) system were isolated from a clinically-isolated Escherichia coli strain HK31. The entire R-M system of EcoHK31l is located in a 2.1 kb fragment. R.EcoHK31l is an isoschizomer of Eael which recognizes and cleaves Y down arrow GGCCR. M.EcoHK31l consists of two polypeptides alpha and beta with sites 309 and 176 aa, respectively. Polypeptide beta is encoded within an alternative reading frame of polypeptide alpha. All the conserved motifs in mC5-MTases can be found in polypeptide alpha except motif IX which is present in polypeptide beta. Polypeptides alpha and beta were separately synthesized in a T7 promoter controlled over-expression system and in vitro methylation occurred only when the two extracts were mixed and thus confirms that two polypeptides are required for methylation.