Changes in ribosomal activity of Escherichia coli cells during prolonged culture in sea salts medium.
The activity of ribosomes from a clinical isolate of Escherichia coli, exposed to starvation for 7 days in sea salts medium, was investigated by measuring the kinetic parameters of ribosomal peptidyltransferase, by using the puromycin reaction as a model reaction. No alterations in the extent of peptide bond formation were observed during starvation. In contrast, a 50% reduction in the kmax/Ks ratio could be seen after 24 h of starvation; an additional 6 days of starvation resulted in a progressive but less abrupt decline in the kmax/Ks value. (kmax is the apparent catalytic rate constant of peptidyl transferase, and Ks is the dissociation constant of the encounter complex between acetyl (Ac)[3H]Phe-tRNA-poly(U)-ribosome and puromycin.) Although the distribution of ribosomal particles remained constant, a substantial decrease in the number of ribosomes per starved cell and a clear decline in the ability of ribosomes to bind AcPhe-tRNA were observed, particularly during the first day of starvation. Further analysis indicated that rRNA in general, but especially 23S rRNA, was rapidly degraded during the starvation period. In addition, the L12/L7 molar ratio decreased from 1.5 to 1 during the initial phase of starvation (up to 24 h) but remained constant during the subsequent starvation period. Ribosomes isolated from 24-h-starved cells, when artificially depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells, regenerated an L12/L7 molar ratio of 1.5 and restored the peptidyltransferase activity to a substantial level. An analogous effect of reconstitution on the efficiency of ribosomes in binding AcPhe-tRNA was evident not only during the initial phase but throughout the starvation period.
Kalpaxis
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DL
Laboratory of Biochemistry, School of Medicine, University of Patras, Greece. DIMKAL@MED.UPATRAS.GR
Karahalios
P
P
Papapetropoulou
M
M
D003470:Culture Media; D004926:Escherichia coli / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006801:Humans; D007700:Kinetics; D010458:Peptidyl Transferases / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D003470:Culture Media; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D012346:RNA, Transfer, Amino Acyl; C053634:RPL7 protein, human; D012269:Ribosomal Proteins; C512844:rplL protein, E coli; C006383:tRNA, N-acetylphenylalanine-; C016700:ribosomal protein L7-L12; D010458:Peptidyl Transferases