CLONING AND ANALYSIS OF TRANSLATIONAL CONTROL FOR GENES ENCODING THE CFR9I RESTRICTION-MODIFICATION SYSTEM

The complete type-II Cfr9I restriction-modification (R-M) system of Ci trobacter freundii strain RFL9, recognizing the DNA sequence CCCGGG, h as been cloned and expressed, and functionally active enzymes have bee n produced in Escherichia coli. Both the methyltransferase (MTase; M.C fr9I) and restriction endonuclease (ENase; R.Cfr9I) were found to be e ncoded on a 2.3-kb cloned fragment in the same transcriptional orienta tion, but differing in translational phases. The last codon (underline d) (ATGA) of the MTase-encoding gene (Cfr9IM) overlaps with the start codon for the ENase-encoding gene (overlined) (cfr9IR). A nucleotide s equence complementary to a predicted Shine-Dalgarno sequence preceding cfr9IR is within this gene. Predicted free energy (Delta G) for forma tion of the mRNA secondary structure involving these complementary seq uences was found to be - 16.1 kcal/mol. Amino-acid sequence homology o f 80% was found between R.Cfr9I and R.XcyI.