Inactivation and reactivation of ribosomal subunits: Amino acyl-transfer RNA binding activity of the 30 s subunit of Escherichia coli

Abstract

The ability of the 30 s ribosomal subunit to bind phenylalanyl-transfer RNA in the cold in response to polyuridylic acid is lost if the subunit is subjected, even transiently, to either of two treatments: (a) the removal of certain specific monovalent cations ( NH + 4, K +, Rb + or Cs +), or (b) the reduction of the Mg 2+ concentration below a critical concentration of about 2 m m. If the depleted cation is restored, the subunit reverts to an active form in a process that is greatly enhanced by heat. Thermally reactivated subunits retain full activity when rechilled, showing that the inactivation and reactivation processes involve changes, presumably conformational, in the subunit itself. Reactivation follows first-order kinetics with respect to the appearance of active subunits, with an Arrhenius activation energy of 26 kcal./mole between 30 °C and 40 °C. On storage at 0 °C, inactive 30 s subunits gradually lose the ability to be reactivated. Part of this loss is due to the oxidation of one or more sulphydryl groups and is prevented or reversed if a sulphydryl reducing agent is included in the storage or the reactivation medium, respectively. Active and inactive 30 s subunits have the same sedimentation coefficient and there is no direct evidence that they differ in conformation. However, two kinds of indirect evidence are in accord with the existence of conformational differences: (a) under appropriate conditions inactive 30 s subunits form dimers sedimenting at 50 s while active subunits do not, and active 30 s subunits associate more readily with 50 s subunits to form 70 s ribosomes; (b) inactive 30 s subunits undergo sulphydryl oxidation much more rapidly than do active ones. Although differing in certain details, the 30 s inactivation and reactivation processes are generally similar to those previously described for the 50 s subunit. Both subunits can exist in active and inactive forms which are easily and reversibly interconverted, suggesting that the structure of the functional ribosome is flexible and easily altered. The interconversions affect a number of ribosomal activities in parallel. It is possible that many previously described phenomena pertaining to ribosomal activity can be interpreted, at least in part, in terms of ribosomal inactivation and reactivation.