Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16 S ribosomal RNA of Escherichia coli. II. The effect of the absence of the methyl groups on initiation of protein biosynthesis

Abstract

The effect of the presence or absence of methyl groups on the N6 atoms of two adjacent adenosines near the 3' end of 16 S rTNA of Escherichia coli on initiation of protein biosynthesis has been studied using wild type (methylated) and kasugamycin-resistant (unmethylated) E. coli ribosomes (see preceding paper (Poldermans, B., Goosen, N., and Van Knippenberg, P. H. (1979) J. Biol. Chem. 254, 9085--9089)). Conditions of pH, temperature, and ionic strength at which binding of fMet-tRNA to ribosomes proceeds maximally are the same for wild type and mutant ribosomes. Mg2+- and factor-dependent dissociation of ribosomes as well as the association of the subunits is also the same for methylated and unmethylated ribosomes. Binding of fMet-tRNA to wild type and to mutant 70 S ribosomes requires the same amount of the three initiation factors. However, optimal fMet-tRNA binding to unmethylated 30 S ribosomes needs more of initiation factor 3 than does binding to methylated 30 S ribosomes, provided that initiation factor 1 is absent. This difference is completely abolished when mutant 30 S ribosomes are methylated using purified methylase from the wild type strain and the methyl donor S-adenosylmethionine.