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

^a Laboratory of Developmental Microbiology, Department of Microbiology, University of Virginia Medical School, Charlottesville, Virginia 22901

ABSTRACT

Two temperature-sensitive mutants (lysS1 and lysS2) of the lysyl-transfer ribonucleic acid synthetase (L-lysine:tRNA ligase [adenosine 5'-monophosphate], EC 6.1.1.6) of Bacillus subtilis have been isolated. Although protein synthesis is inhibited in both mutants at the restrictive temperature (42 to 45 C), the mutants remain viable in a minimal medium. In comparison with the wild-type lysyl-tRNA synthetase, the L-lysine-dependent exchange of [³²P]pyrophosphate with adenosine 5'-triphosphate (ATP) for both mutant enzymes is decreased. The lysS1 enzyme is completely defective in the ATP-dependent attachment of L-lysine to tRNA, whereas the lysS2 enzyme has 3- to 10-fold reduced levels of this activity. Temperature-resistant transformants have wild-type enzyme levels, whereas partial revertants to temperature resistance have varied levels of enzyme activity. The attachment and exchange activities of the lysS2 enzyme are more heat labile in vitro than the wild-type enzyme, as is the attachment activity of a partial revertant of the lysS1 mutant. The lysS1 and the lysS2 lysyl-tRNA synthetases have higher apparent K_m values for lysine and ATP, in both the activation and the attachment reactions. The lysS2 enzyme has a V_max for tRNA^lys one-third that of the wild-type enzyme. Molecular weights of 150,000 for the wild-type and lysS2 enzymes and 76,000 for the lysS1 enzyme were estimated from sedimentation positions in sucrose density gradients assayed by the ATP-pyrophosphate exchange activity. We propose that the two mutations (lysS1 and lysS2) directly affect the sites for exchange activity, but indirectly alter attachment activity as a consequence of defective subunit association.

¹ Present address: Department of Biological Chemistry, University of Illinois Medical Center, Chicago, Ill. 60612.