Protein synthesis kinetics with ribosomes from temperature-sensitive mutants of Escherichia coli

Abstract

The kinetics of MS2 ribonucleic acid (RNA) directed protein synthesis have been investigated at seven temperatures between 30 and 47 degrees C by using ribosomes isolated from a wild type strain and seven temperature-sensitive mutants of Escherichia coli. The amount of MS2 coat protein formed at each temperature was determined by gel electrophoresis of the products formed with control ribosomes. With ribosomes from each of the mutant strains, the activation energy required to drive protein synthesis below the maximum temperature (up to 40 degrees C) was increased relative to the control (wild type) activity. Preincubation of the ribosomes at 44 degrees C revealed the kinetics of thermal inactivation, with ribosomes from each of the mutants having a half-life for inactivation less than that of the control ribosomes. A good correlation was observed between the relative activity of the different ribosomes at 44 degrees C and their relative rate of thermal inactivation. Mixing assays allowed the identification of a temperature-sensitive ribosomal subunit for each of the mutants. Defects in one or more of three specific steps in protein synthesis (messenger RNA binding, transfer RNA binding, transfer RNA binding, and subunit reassociation) were identified for the ribosomes from each mutant. The relationship between temperature sensitivity and protein synthesis in these strains is discussed.