CONDITIONAL LETHAL MUTATION IN AN ESCHERICHIA-COLI STRAIN WITH A LONGER CHEMICAL LIFETIME OF MESSENGER-RNA

We have analysed an Escherichia coli temperature-sensitive mutant with altered messenger RNA stability, and it was found that: (1) the unstable fraction of pulse-labelled RNAs decayed with a half-life at 42 °C of about two minutes in the parent strain PA3092; the half-life was 11 to 12 minutes in the mutant HAK75. Puromycin enhanced the decay rate about twofold in both PA3092 and HAK75; the addition of chloramphenicol inhibited the degradation significantly in both strains. The rate of ribosomal RNA accumulation in the mutant cells at 42 °C did not differ from that in the wild-type cells. (2) Sedimentation analysis by sodium dodecyl sulphate/sucrose density-gradient centrifugation of bulk mRNA as well as tryptophan mRNA of the wild-type strain showed the expected rapid reduction in the size and level of those mRNA molecules at three minutes and five minutes respectively, after addition of rifampicin at 42 °C. In contrast, the cells of HAK75 retained almost full-length trp mRNA and bulk mRNA at 5 to 12 minutes after the addition of rifampicin at 42 °C, even though the total level of radioactivity in the mRNA fraction had decreased to about 60 to 75% of the initial activity. (3) Even though mRNA molecules were chemically protected at the non-permissive temperature in the mutant, the functional decay of both β-galactosidase and tryptophan synthetase occurred at a rate comparable to that in the parental strain. (4) We isolated temperature-resistant revertants from the mutant at a frequency of 5 × 10-8, and these revertants (TR1 and TR2) had the normal decay rate of unstable RNA. © 1979.