SUBSTRATE REQUIREMENTS FOR ERMC' METHYLTRANSFERASE ACTIVITY

ErmC' is a methyltransferase that confers resistance to the macrolide- lincosamide-streptogramin B group of antibiotics by catalyzing the met hylation of 235 rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmC' was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Studies of substrate requirements of ErmC' ha ve shown that a 262-nucleotide RNA fragment within domain V of B. subt ilis 235 rRNA can be utilized efficiently as a substrate for methylati on at A-2085. Kinetic studies of the monomethylation reaction showed t hat the apparent K-m of this 262-nucleotide RNA oligonucleotide was 26 -fold greater than the value determined for full-size and domain V 235 rRNA. In addition, the V-max for this fragment also rose sevenfold. A model of RNA-ErmC' interaction involving multiple binding sites is pr oposed from the kinetic data presented.