The project is designed to extend previous studies concerning the characterization of a rRNA-modification enzyme, namely rRNA-adenine (N6-) methylase of E. coli strain K12, in areas of (a) purification of the methylase(s), (b) elucidation of the mechanism of site-selection, and (c) determination of the mode of regulation of enzyme synthesis and modulation of the activity. Previous studies demonstrated that the synthesis of the methylase is under metabolic control, the ratio of methylase activity/RNA being constant regardless of growth rate, with indications that the enzyme activity may be modulated. The potential effector, ppGpp, will be isolated and employed in in vitro studies to determine whether it alters the activity and the mechanism by which the modulation is accomplished. In vivo studies will be utilized to determine (a) the consequences to rRNA modification and ribosomal assembly when the methylase/RNA ratio is altered and (b) the mechanism by which the p-rRNA is "rescued" when the cell restores this ratio. A homologous methylase - rRNA (nonmethylated) system will be employed to determine whether two distinct methylases exist, each specific for the synthesis of one of the two N6-methyladenine moieties in 23s rRNA. One site (early-site) is expected to be methylated in the absence of ribosome assembly, while the second (late-site) may require prior addition of ribosomal proteins. The sequence surrounding each site will be determined. The ability of ribosomal proteins to inhibit and/or facilitate the methylation of both the early- and late-sites will be investigated.