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J Bacteriol. 1973 June; 114(3): 988-998
Copyright © 1973 American Society for Microbiology. All Rights Reserved.

Ribosomal Ribonucleic Acid-Adenine (N⁶-) Methylase of Escherichia coli Strain B: Ionic and Substrate Site Requirements

^a Department of Biochemistry, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103

ABSTRACT

These investigations are concerned with the ionic and substrate-site requirements of ribosomal ribonucleic acid (rRNA)-adenine (N⁶-) methylase of Escherichia coli B. The methylase was essentially inactive in solutions of low ionic strength. The addition of MgCl₂ (optimal at 5 mM) or; to a lesser degree, KCl (optimal at 45 mM) stimulated the rate of methylation; the combination of MgCl₂ and KCl stimulated methylation to an extent equivalent to the sum of the stimulation of each acting alone. The extent of nonspecific binding of the methylase to rRNA decreased as the ionic strength of the solution increased. In the absence of ions, dimethylsulfoxide (DMSO), a nucleic acid denaturing agent, had little influence on the rate of methylation; however, DMSO plus KCl synergistically increased both the rate and the extent of methylation to a greater degree than the combination of Mg²⁺ plus K⁺. NH₄⁺ was less effective than K⁺, and the divalent Mg²⁺ offered little stimulation. Monovalent anions (acetate, nitrate, and chloride) were equally effective, whereas divalent SO₄^2– was decidedly inhibitory. The appropriate ionic milieu of mono- and divalent cations was required to provide the appropriate conformation of the rRNA and to facilitate specific interactions of the methylase and its recognition sites in the rRNA, while decreasing nonspecific ionic binding of the methylase to rRNA. DMSO may facilitate methylation by increasing the number of substrate sites exposed in single-stranded regions of the rRNA. Nonmethylatable rRNA species served as competitive inhibitors, whereas the polyanions deoxyribonucleic acid, transfer RNA, and polyadenylic acid were inactive. Micrococcus lysodeikticus and Bacillus subtilis rRNA, methylated by the methylase, each contained two distinct heptanucleotides containing newly synthesized 6-methyladenine moieties. The data are consistent with the view that E. coli strain B possesses two species of rRNA-adenine (N⁶-) methylases, each of which recognizes a specific adenine moiety in a unique pentapurine nucleotide sequence in a single-stranded region of rRNA.

¹ Present address: Laboratory of Molecular Biology, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.