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Publisher Summary The many demonstrations that the Escherichia coli (E. coli ) rne gene product (RNase E) is involved in messenger RNA (mRNA) decay have given real impetus to the study of this unusual protein's properties and role. The recent attention given to the polyadenylylation of bacterial mRNAs and the discovery that polyadenylylation plays a role in the turnover of an E. coli plasmid-specific RNA show that bacterial mRNA decay has similarities to the eukaryotic process. Functional decay of a messenger refers to the inactivation of a messenger's template activity, whereas chemical decay refers to the degradation of the mRNA itself. Functional decay, as a process distinct from the cleavages that initiate degradation, has been demonstrated for only a few mRNAs. Specific messengers have half-lives that range from about 0.5 to 20 minutes. The rates of decay of many messengers are actively regulated, thus, affecting the yields of the proteins synthesized. Unique to bacteria, portions of polycistronic mRNAs may decay at different rates, independent of the size of the segment or its position in the transcript. Overall, the decay of mRNA in bacterial cells is a major metabolic function. The turnover of mRNA constitutes about half of the cells' RNA synthesis; the actual amount is related to their growth rate.

Publisher Summary This chapter describes the purification procedure for phosphoribosylglycinamide synthetase from Aerobacter aerogenes organism. Phosphoribosylamine: glycine ligase [ADP] (PRG) synthetase catalyzes in the de novo path of purine biosynthesis in bacteria as well as higher organisms. The chapter discusses the preparation procedure from Aerobacter aerogenes or, according to the current classification of the original strain, Klebsiella pneurnoniae. Two assays are described. The colorimetric assay is recommended for enzyme purification. It is fast, sensitive, and works well with crude enzyme preparations. The second––radioisotopic assay––is more readily quantitated. It does not work well with crude enzyme preparations. The measurement is carried out in two steps: the formation of PRG and the PRG assay. The purification is carried out at 3° unless otherwise stated. Crude cell extracts are stable for one day of storage on ice, and enzyme at the intermediate stages of purification is stable for several days.

Bacteriophage T4 possesses a cluster of tRNA genes which are expressed as immediate early functions of phage infection. In vitro the tRNAs appear transcribed polycistronically from a single promoter. Transcription from this site is made resistant to rifampicin inactivation by the addition with rifampicin of ATP, UTP and CTP. This stabilization, which is a common characteristic of several of the T4 early promoters, allows one to separate the initiation and elongation steps of gene expression and facilitates the study of promoter function. The tRNA genes appear to be preferentially transcribed among those operons initiated in the presence of ATP, UTP and CTP. We have studied initiation using a multistep procedure in which we assume only 1 polymerase per ATP initiated operon becomes rifampicin resistant. We find that the RNA polymerase concentration needed to support a half-maximal tRNA synthesis is 5 fold lower than that needed to support half-maximal total RNA synthesis. This difference appears to be due to differences in the affinities of the promoters for polymerase. The temperature of activation of the tRNA promoter is similar to or only slightly lower than that of other ATP initiated promoters, that is, about 12° at 0.05 M KC1 concentration. This suggests that the difference does not lie in the ease in which the open complex is formed, but rather in the recognition of the promoter site itself.

From Eco Rl restriction fragments of T4 DNA, several fragments have been purified to apparent homogeneity, each of which contains the cluster of eight tRNA genes carried by the bacteriophage. Under our conditions these fragments are approximately 10 fold less active on a molar basis in the in vitro synthesis of the tRNAs than the non-restricted template, yet they appear as normal templates in other respects, e.g., the synthesis of tRNAs from proximal and distal regions of the cluster. In this report the possibility that this apparent low template efficiency may be due to an adversely high RNA polymerase/DNA ratio in the reactions was examined in experiments with intact T4 DNA. An inhibitory effect of RNA polymerase was seen acting on the elongation reaction of transcription. This inhibition appears to be due to the binding of polymerase to the template. It can be reversed by the addition of poly rI or heparin to the mixtures following the initiation of transcription. At appropriately low levels of RNA polymerase, the isolated fragments are transcribed selectively. The product formed contains up to six large asymmetric species indicating that the fragments bear at least one discrete initiation site for the tRNA operon.