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Ca²⁺ Ions Inhibit Messenger Ribonucleic Acid Degradation, but Permit Messenger Ribonucleic Acid Transcription and Translation in Deoxyribonucleic Acid-coupled Systems from Escherichia coli

From the ¹ From the Department of Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110

DNA-directed formation of RNA and protein was compared in subcellular systems in the absence or presence of Ca²⁺ ions. In the absence of Ca²⁺, messenger RNA was unstable, and protein and RNA accumulation tended to stop after incubations of 10 to 15 min at 37°. In contrast, in the presence of Ca²⁺ mRNA was stabilized almost completely. As a result, the net accumulation of RNA and protein continued for 20 to 45 min at 37°, and could be 3-fold greater, even though the rates of formation are lower.

The fate of the RNA in the presence and in the absence of Ca²⁺ was detailed after limitation of synthesis by rifampicin, in DNA-coupled systems modified from those of Lederman and Zubay (Lederman, M., and Zubay, G. (1967) Biochim. Biophys. Acta 149, 253) or Gold and Schweiger (Gold, L. M., and Schweiger, M. (1971) Methods Enzymol. 20, 537). In the presence of Ca²⁺, little or no acid-soluble material was released during 30 min from RNA formed on T4, Escherichia coli, 80, or DNAs, and T4-specific mRNA extracted at various times and analyzed by acrylamide gel electrophoresis showed no detectable cleavages to smaller sized chains.

The implication that a number of ribonucleases are directly inhibited by Ca²⁺ ions was confirmed for two purified E. coli enzymes, the endonuclease RNase III and the exonuclease RNase II. Thus, Ca²⁺ ions provide an effective way to dissociate in vitro the physiological coupling of mRNA formation and translation from its degradation.

In the absence of Ca²⁺, a number of features of mRNA metabolism in vivo could be reproduced. Chemical decay of E. coli and T4-specific mRNA was studied. As in vivo, decay of both types of mRNA was exponential. Also, the half-lives (2.25 min for E. coli and 4.45 min for T4 mRNA) were of the same magnitude and in the same ratio as in vivo. Furthermore, the response of mRNA metabolism to antibiotics was comparable to that in vivo; when protein synthesis was blocked by the addition of chloramphenicol, new mRNA was protected against decay, but when puromycin was used to block protein synthesis, mRNA still degraded rapidly.

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