Properties and Developmental Roles of the Lysyl- and Tryptophanyl-Transfer Ribonucleic Acid Synthetases of Bacillus subtilis : Common Genetic Origin of the Corresponding Spore and Vegetative Enzymes

Abstract

The lysyl-transfer ribonucleic acid synthetase (LRS) and tryptophanyl-transfer ribonucleic acid synthetases (TRS) (L-lysine :tRNA ligase [AMP], EC 6.1.1.6; and L-tryptophan: tRNA ligase [AMP], EC 6.1.1.2) have been purified 60-and 100-fold, respectively, from vegetative cells and spores of Bacillus subtilis. There are no significant differences between the corresponding spore and vegetative enzymes with respect to their elution characteristics from columns of phosphocellulose or hydroxylapatite, their molecular weight (-~130,000 for LRS and -87,000 for TRS as determined by gel filtration), their kinetic constants for substrates (in the amino acid-dependent adenosine triphosphate-pyrophosphate exchange reaction), and the kinetics of inactivation by heat and by antibody. The Mg2+ requirement for optimal enzyme activity of the corresponding spore and vegetative enzyme differ slightly. Mutants having defective (temperature sensitive) vegetative LRS or TRS activities produce spores in which these 70 on August 1, 2020 by guest

Defects of Two Temperature-Sensitive Lysyl-Transfer Ribonucleic Acid Synthetase Mutants of Bacillus subtilis

“…The] from the equation of Martin and Ames (24). L-lysine These values agree with the values of -87,000 class II and -130,000 obtained by gel filtration (34). growth, b…”

“…The material comprising the void volume of the Bio-Gel P-6 column was further purified on a column of phosphocellulose (1 by 2 cm) equilibrated with buffer A plus 0.1 M KCl. The column was washed with 4 column volumes of equilibrating buffer, and the sample was eluted with buffer A plus 0.6 M KCl (34). This resulted in an eightfold purification for LRS over the activity of the Bio-Gel fraction.…”

“…LRS assays. LRS activity was determined by the ATP-dependent attachment of L-[14C ]lysine to tRNA, or by the L-lysine-dependent exchange of [32P]pyrophosphate with ATP (33,34). Unless indicated, the reaction incubation periods (at 25 C) were 10 min and 30 Km determinations.…”

“…Extracts for the determination of kinetic constants in the attachment assay were prepared by method (ii). Reactant concentrations were as described previously (34), except for the substrate in question, which was varied.…”

Comparative aspects of development and differentiation in actinomycetes.

“…The scarce information on transcriptional and translational controls available with actinomycetes will be presented in the following section, as well as in the one dealing with secondary metabolism. Mounting evidence stemming from the work with sporulating bacteria (275,441,480) suggests that at least some spore proteins might be products ofvegetative genes. Besides emphasizing the importance of post-transcriptional controls, these facts might also reflect the economy of information stored in chromosomal genes.…”