Kinectics of beta-galactosidase synthesis in Escherichia coli at 5 C

Abstract

The defect in protein synthesis that is observed in Escherichia coli after transfer to low temperature was studied. For the enzyme ,8-galactosidase, the elongation reactions of transcription and translation can take place slowly but normally at 5 C. The time necessary to complete the coupled synthesis of the ,8-galactosidase messenger ribonucleic acid and polypeptide chain was found to be about 80 min at 5 C. From this result and from the known length of the ,-galactosidase monomer, it is possible to calculate that at 5 C one amino acid is added to the growing polypeptide chain every 4 s. The initiation of transcription of the d-galactosidase messenger is inhibited after transfer to 5 C. This fact alone, however, cannot account for all of the phenomena observed at 5 C, because a given amount of messenger yields less enzyme at 5 C than it does at 37 C. Furthermore, in cells induced for short periods at 37 C, the capacity to synthesize fB-galactosidase after transfer to 5 C was found to accumulate linearily with the square of the time of induction. Two alternative models could account for these data. If all ribosomes that initiate translation at 37 C yield complete ,-galactosidase polypeptide chains at 5 C, then an inhibition of translation initiation after transfer to 5 C must be invoked to explain the results. If, on the other hand, a substantial portion of the ribosomes that initiate translation at 37 C do not yield complete A-galactosidase polypeptides at 5 C, then intracistronic polarity could account for the data, and there is no need to invoke an inhibition of translation initiation at 5 C.The minimum temperature that permits sustained growth of Escherichia coli is in the vicinity of 7.8 C (22). Das and Goldstein (6) studied macromolecular syntheses after a transfer of exponentially growing cells of E. coli from 37 to 0 C. They observed that the incorporation of radioactive leucine into protein proceeded at a slowly decreasing rate over a period of 4 h, but that incorporation of radioactive uridine into ribonucleic acid (RNA) continued in a linear fashion during this same period. Based on this and other indirect evidence, they inferred that initiation of translation at the 5' end of messengers is blocked at 0 C, but that polypeptide chain elongation can continue. In addition, it has been shown (1, 4, 6) that low-temperature incubation of E. coli leads to polysomal run-off, with the consequent accumulation of free 70S ribosomes (1, 4). Friedman et al. (9) concluded that f2 viral coat protein is completed but not initiated after transfer from 37 to 6 C. They (9) also observed that initiation of polypeptide chain synthesis from E. coli messengers is severely restricted at 4 C; however, it was not clear from their experiments whether this restriction was due to a block at the level of initiation of transcription or of translation.Based on these results, it seemed possible that an uiusual translational control mechanism might explain the limited capacity of E. coli cells for protein synthesis at low temperature. ...

Effects of low temperature on in vivo and in vitro protein synthesis in Escherichia coli and Pseudomonas fluorescens

“…coli and P. fluorescens whole-cell systems and in vitro protein-synthesizing systems [directed by poly(U), MS-2 viral RNA, or endogenous polysomes] were tested, demonstrate that: (i) polypeptide elongation is inhibited to nearly the same extent in both organisms over the range 0 to 25°C, and (ii) elongation is able to proceed in the E. coli systems only for the length of time necessary for the polysomes or preloaded ribosomes to run off the mRNA. The conclusion that polypeptide elongation is not blocked at low temperature is supported by the work of others (1,5,9,29).…”

“…However, if an early event in the process of initiation was largely inhibited, but not completely blocked at 50C (i.e., limiting the rate of initiation at 50C), 70S monosomes would be a minor component of the 70S ribosome population because elongation proceeds at a greater rate than initiation at 50C. Data reported by others (1,4), preliminary data (to be published elsewhere) obtained in our laboratory, and the data shown in Fig. 8 (above), which shows that a significant amount of the 70S particles that accumulate at 50C can be dissociated by high-speed centrifugation (and possibly RNase), indicate that the latter view may be the case.…”

“…The minimum temperature for growth of Escherichia coli is in the vicinity of 7.80C (27). A number of workers have studied the effects of low temperature on in vivo (1,4,5,8,9) and in vitro (7,30) protein synthesis by E. coli and have linked the inability to grow to the inhibition of protein synthesis at temperatures below 7.80C. In a recent study (to be published elsewhere) comparing the effects of near-00C temperatures on a number of physiological and molecular processes in a variety of marine and terrestrial bacteria, we demonstrated that when mesophilic bacteria were shifted to subminimal growth temperatures (50C), protein synthesis was the process which was most severely inhibited, whereas the psychrotrophic and psychrophilic strains synthesized protein at 50C rates which corresponded to their 50C growth rates.…”