Stability of Ribosomes and Ribosomal Ribonucleic Acid from Bacillus stearothermophilus

Abstract

After heating at 65 C, ribosomes isolated from Bacillus stearothermophilus were strikingly more heat-stable than comparable preparations from Escherichia coli when tested for ability to support polyuridylic acid-directed phenylalanine incorporation at 37 C. The stability of ribosomes was also determined by measurements of hyperchromicity at 259 m,u while heating them from 25 to 90 C. In standard buffer containing 0.01 M Mg+, the Tm (temperature at the midpoint of total hyperchromicity) of E. coli and B. stearothermophilus ribosomes was 71 and 81 C, respectively. In a magnesium-free buffer, the Tm of E. coli and B. stearothermophilus ribosomes was 44 and 64 C, respectively. Putrescine (0.01 M) was more effective in stabilizing ribosomes from B. stearothermophilus than those from E. coli. Spermidine (0.001 M), on the other hand, was more effective in stabilizing ribosomes from E. coli than those from B. stearothermophilus. Melting curves of total ribosomal ribonucleic acid (rRNA) from E. coli and B. stearothermophilus revealed Tm values of 50 and 60 C, respectively. Putrescine stabilized thermophile rRNA, but had no effect on E. coli rRNA. Sucrose density gradients demonstrated that thermophile 23S ribonucleic acid was degraded during storage at-20 C; the 23S component from E. coli was stable under these conditions. The results are discussed in terms ofthe mechanism of ribosome heat stability and the role of the ribosome in governing the temperature limits for bacterial growth.

Physiology of Growth at High Temperatures

“…3). These and similar data obtained by Pace & Campbell (1967), Stenesh & Yang (1967), Stenesh & Holazo (1967) and Friedman et al (1967) makes improbable the possibility that the increased heat stability of thermophile ribosomes arises from the ribosomal RNA per se. Saunders & Fig.…”

“…coli ribosomes lost 90% of their activity after 5 min at 65", similar treatment of B. stearothermophilus ribosomes led to a slight increase (c. 10%) in activity. Freidman & Weinstein (1966) and Friedman, Axel & Weinstein (1967) have reported similar observations. Friedman et al (1967) have tested heated ribosome suspensions of B. stearothermophilus and E. coli for their ability to support polyuridylic acid-directed phenylalanine incorporation at 37".…”

“…Freidman & Weinstein (1966) and Friedman, Axel & Weinstein (1967) have reported similar observations. Friedman et al (1967) have tested heated ribosome suspensions of B. stearothermophilus and E. coli for their ability to support polyuridylic acid-directed phenylalanine incorporation at 37". After 5 min a t 65", 94% of the initial activity was retained by the thermophilic ribosomes, whereas only 21 % was retained with E. coli ribosomes.…”

“…They are also consistent with the results obtained by Bubela & Holdsworth (1966a, b ) on protein synthesis by whole cells and cell fractions of B. stearothermophilus. Heat stability of ribosomal RNA and ribosomes as determined by thermal melting profiles Ribosomes isolated from B. stearothermophilus undergo thermal denaturation at temperatures markedly higher than those obtained from E. coli, as judged by measurement of the hyperchromicity a t 260 m p with increasing temperatures (Mangiantin&, Tecce & Toschi, 1962;Mangiantini, Tecce, Toschi & Trontalance, 1965;Saunders & Campbell, 1966;Pace & Campbell, 1967;Stenesh & Yang, 1967;Stenesh & Holazo, 1967;Friedman et al, 1967). To determine if the greater stability of B. stearothermophilus ribosomes was due to the presence of an unusual ribosomal RNA species Saunders & Campbell (1966) studied the ribosomal RNA of this organism.…”

The Role of Ribosomal Conformation in Protein Biosynthesis

“…It is per-tinent that misreading of the code can also be brought about by the presence of high levels of Mg2+ [4, 30,31] or polyamines [31]. Ribosomes under these conditions are stabilized from thermal denaturation [32]. Stabilization of ribosomal structure in this way may thus be analagous to the "tightening" phase induced a t high streptomycin/ribosome ratios, under which conditions miscoding occurs.…”