Polyribosome metabolism in Escherichia coli: II. Formation and lifetime of messenger RNA molecules, ribosomal subunit couples and polyribosomes

Abstract

During a continuous label with [ 3H]uracil of fragile Escherichia coli cells in exponential growth, all or very nearly all the hybridizable mRNA of the cells can be observed in polyribosomes during zonal sedimentation in sucrose gradients. In contrast, all the newly formed rRNA appears to be free of polyribosomes until it is part of complete ribosomal subunits. The separation of the two major classes of newly formed RNA permits the direct observation of the growth with time of mRNA molecules, of ribosomal subunits and of polyribosomes. A complete chain of ribosomal RNA forms in one to two minutes; completion of either a 30 s or 50 s ribosome requires a minimum of about five minutes more. The specific activities of free ribosomal subunits and those that are present in polyribosomes increase at identical rates, consistent with very rapid exchange of the two classes. Before any new subunits are finished, the 3H-labeled RNA in polyribosomes is the cellular mRNA, which can therefore be extracted and observed separated from ribosomal RNA. Assuming that their sedimentation rates in sucrose gradients are related to molecular weights as are those of ribosomal rRNA and that no degradation occurs during extraction, most of the molecules of mRNA are large enough to code for only one or two protein chains of 30,000 molecular weight. The kinetics of entry of all the new mRNA into polyribosomes is in agreement with the view that a polyribosome forms as the corresponding messenger RNA is synthesized, over a period of at least a minute. 3% of the total cellular RNA in these cultures (doubling time 120 minutes) is mRNA, with an average chemical lifetime of 11 to 12.5 minutes.