In vivo reassembly of 30-S ribosomal subunits following their specific destruction by thermal shock

Abstract

When Escherichia coli cells suspended in 0.25 M potassium phosphate, pH 7.2 are heated at 48°C for 25 min the 30-S ribosomal subunits are completely destroyed while the 50-S subunits remain intact and active. On transfer to Davis minimal medium with shaking at 30°C, the bacteria recover from the shock and reassemble new 30-S subunits which consist of newly synthesized 16 S rRNA and preexisting 30-S ribosomal proteins. Kinetic studies show that the RNA synthesis capability of the recovering bacteria attains a level similar to untreated cells within 30 min of incubation; by contrast, their protein synthesis capability in terms of l-[ 3H]leucine incorporation is neglible for as long as 90 min, at which time it begins to increase sharply and reaches the control levels after an additional 60 min. Sedimentation analyses of cell extracts show that the new subunits begin reforming after about 45 min of incubation and the sedimentation profile acquires a normal appearance at 90 min. Various kinds of evidence indicate that the recovery process involves at least 45% of the original bacteria, including studies of the various types of rRNA species, the composition of ribosomal proteins in the new subunits, and the nature of the extended lag in recovery before the onset of protein synthesis. Both 16 S and 23 S rRNA are accumulated, albeit with a predominance of the 16 S species. After 90 min the ribosomal proteins S1 and S21 are still unpresented in the reassembled 30 S particle while S2 and S10 are absent altogether. This is in agreement with the reconstitution experiments of Nomura and Held (Nomura, M. and Held, W.A. (1974) in Ribosomes (Nomura, M., Tissieres, A. and Lengyel, P., eds.), pp. 193–223, Cold Spring Harbor Laboratory, New York), in which S10 and S21 are among the last to be attached. Since protein synthesis begins after 90 min, it is likely that a small fraction of the reassembled 30-S subunits already contain the whole set of proteins while most of them still do not. The gross time required for in vivo reassembly can be estimated by the onset of l-[ 3H]leucine incorporation. In the presence of methionine this incorporation begins about 30 min earlier than in its absence. The effect of methionine is due to the thermolability of homoserine trans-succinylase. The net time required for the reassembly is estimated to be about 30 min.