Measurement of the unstable RNA in exponentially growing cultures of Bacillus subtilis and Escherichia coli

A new technique for measuring the rate of turnover and quantity of unstable RNA in exponentially growing cells has been developed which escapes the interpretive difficulties of earlier methods. It depends upon the fact that an unstable RNA fraction has easily measurable effects upon the kinetics of labelling of GTP; before the GTP pool can become fully labelled, unlabelled nucleotides present as GTP or in unstable RNA must be washed into stable RNA. (There is no equilibration with guanine in the medium.)Consequently, if the uptake of label into GTP is rapid, with only the delay predicted from the experimentally measured GTP pool size, then it is possible to conclude that there is little or no unstable RNA. In Bacillus subtilis, however, the GTP pool is labelled very slowly which, with other data, indicates a large amount of unstable RNA (9% of the guanine residues of the cell in unstable RNA). Similar measurements of the rapidity of GTP labelling in Escherichia coli indicate that there is much less unstable RNA (about 3%) in this organism.The curve expressing GTP specific activity as a function of time is shown to be the sum of two exponential terms with very different time constants. From the relative sizes of the two exponential terms, it is possible to estimate the rate of turnover of the unstable RNA. The result is weighted most heavily for the slow components of the decaying messenger, and thus it sets an upper limit to the true average decay time for the unstable RNA. This decay time (upper limit) was three minutes for the unstable RNA in B. subtilis grown at 37 °C with a doubling time of 115 minutes and six minutes when the cells were grown at 30 °C with a doubling time of 225 minutes. For E. coli (growing at 30 °C with a doubling time of 66 minutes) the decay time measured in this way was about four minutes. Thus the decay times as measured in these experiments are the same order of magnitude as have been observed by measuring the decay of pulse-labelled RNA and the decay of protein synthetic capacity after the addition of actinomycin. The various estimates of the decay rate of messenger RNA in bacteria are discussed in terms of the possible theoretical ambiguities in the interpretation of each.