David Elson

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 Mg2+ 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.

Latent enzymic activity of a ribonucleoprotein isolated from Escherichia coli

Abstract A ribonucleoprotein preparation isolated from E. coli possesses both RNase and DNase activity. Evidence is presented which indicates that the enzymes are tightly bound to the nucleoprotein and are probably not contaminants. Essentially all of the RNase activity of the initial crude cell extract appears to be associated with the nucleoprotein. Both enzymes are latent, in that the intact nucleoprotein shows neither RNase nor DNase activity. Enzymic activity appears under conditions where the nucleoprotein structure is destroyed. The possible significance of the presence and latency of the enzymes is discussed.

Inactivation and reactivation of ribosomal subunits: The peptidyl transferase activity of the 50 s subunit of Escherichia coli

The ability of the 50 s ribosomal subunit to catalyze the peptidyl transferase reaction is absolutely dependent on the continued presence of certain monovalent cations in the ribosomal medium. This ability is maintained as long as one of these cations is present and is lost if the cation is removed. The effective cations are NH4+ ≧ Rb+ > K+ > Cs+. Na+ and Li+ are ineffective. Activity is restored, with the same order of effectiveness, if an appropriate cation is added back. Although at high salt concentrations there may be a slow reactivation in the cold, heat greatly accelerates the process at all salt concentrations. Although reactivating cations are present in the peptidyl transferase assay medium, inactive ribosomes remain inactive if the assay conditions do not promote reactivation; i.e. if the assay is performed in the cold. This shows that the effect is on the ribosome and not on the assay. The ribosome can exist in two different states, active and inactive, and can be brought from either state to the other. The rate of reactivation increases markedly with rising temperature or monovalent cation concentration. Reactivation is also accelerated by the presence of aliphatic alcohols in the medium and by substrate, fMet-tRNA, provided that the substrate is actually bound to the ribosome during the heat treatment. Reactivation of ribosomes follows first-order kinetics under a wide variety of conditions. The energy of activation of the process is 40 to 50 kcal./mole below 30 °C but becomes less at higher temperatures. Inactive subunits lack no essential components and have the same sedimentation pattern and coefficient as active ones. However, it seems likely that the interconversion between the active and inactive form involves a conformational change. It is not known if the inactivation-activation process occurs in vivo; the possibility is discussed.

[40] The inactivation and reactivation of Escherichia coli ribosomes

Publisher Summary When E. coli ribosomes or either of their subunits are assayed under certain conditions for any of a number of biological activities, they can be shown to exist in one of several different states: active, inactive, or partially active. The state is determined by the past treatment of the ribosome. These states are reversibly inter convertible and ribosomes can be brought from one to another by relatively mild treatments. Among the factors that influence these inter conversions in vitro are temperature, the ionic environment, and interactions between subunits or between the ribosome and certain other macromolecules that participate in protein synthesis. 70 S, 50 S, and 30 S ribosomes all undergo these inter conversions, although the characteristics of the process may vary somewhat in each case.

Interconversions between inactive and active forms of ribosomal subunits

We have previously reported that the peptidyl transferase activity of the 50 S subunit of E. coli ribosomes is lost if the ribosomes are exposed to media lacking NH: and K’. Activity can be restored, but this requires both (a) the readdition of one of these ions, and (b) heat. The heat requirement is discerned only if the ribosomes are assayed at O’, where they are active only if they were previously heated in the presence of NH: or K+ [ 11. We have now studied a specific function of the 30 S subunit, the non-enzymatic binding of phenylalanyltRNA directed by poly U and assayed at 0”, and have found a similar inactivation and reactivation, with similar effects of specific monovalent cations and heat. In addition, the 30 S subunit is inactivated if the M

A convenient procedure for preparing transfer ribonucleic acid from Escherichia coli

+ concentration is lowered to 1 mM (as is commonly done to dissociate 70 S ribosomes), even if NH: is present. It is of particular interest, however, that ribosomes that have been inactivated toward non-enzymatic binding are active at 0’ in the enzymatic binding reactions mediated by initiation factors or transfer factor T, even if they have not been previously heated.

The location of ribonuclease in Escherichia coli

Abstract A procedure is described for the isolation of transfer RNA (tRNA) from Escherichia coli . The method is a combination of several known techniques. tRNA is extracted from whole cells with phenol, stripped of attached amino acids at pH 8, preferentially solubilized with 2 M LiCl, and reprecipitated with (NH 4 ) 2 SO 4 . The key step is the use of 2 M LiCl, which separates ribosomal RNA from tRNA rapidly and completely. The method is simple and yields tRNA as active as the best preparations that we have obtained with other methods.

Correlation between the peptidyl transferase activity of the 50 s ribosomal subunit and the ability of the subunit to interact with antibiotics

Abstract The RNAase of Escherichia coli appears to be located entirely in the ribosomes. Within the ribosomes, the enzyme is concentrated in the 30-S particle and is absent from the 50-S particle. Implications of these findings are discussed.

Preparation and properties of a ribonucleoprotein isolated from Escherichia coli

Abstract When the peptidyl transferase activity of the 50 s ribosomal subunit is abolished and subsequently restored by mild inactivating and reactivating treatments, the ability of the subunit to interact with the antibiotics erythromycin, chloramphenicol and sparsomycin is also abolished and restored in parallel. This indicates that the antibiotics interact either with the peptidyl transferase site or with other sites that undergo conformational changes during the inactivation and reactivation of the ribosomal peptidyl transferase.

Ribosome activation and the binding of Dihydrostreptomycin: Effect of polynucleotides and temperature on activation☆

Abstract A ribonucleoprotein has been isolated from exponentially growing cells of E. coli by a precipitation technique. It contains 60–65% RNA by weight and has a hyperchromic effect of 50–60%. Its stability and electrophoretic and ultracentrifugal behavior are discussed. It differs from the deoxyribonucleoproteins in that the protein moiety does not seem to be basic, and the RNA and protein moieties appear to be linked together largely by hydrogen bonds. The RNA moiety is digested by pancreatic RNase.