In vitro Transcription of Escherichia coli Ribosomal RNA Genes

Abstract

THE ribosomal cistrons of Escherichia coli comprise only 0.2 to 0.4% of the genome and yet in exponentially growing cultures 40% of the instantaneous rate of RNA synthesis can be ribosomal RNA^1,2. These genes are under stringent control, which means that they are not expressed in wild type (rel⁺) cultures starved for amino-acids³. Travers et al.^4,5 postulated that a protein factor, called ψr, is a positive regulator of the rRNA genes, responsible for both their preferential synthesis and their turn-off during amino-acid starvation. This appealing hypothesis rests on their report that purified RNA polymerase synthesizes no detectable rRNA from E. coli DNA. They concluded that addition of ψr (a protein factor found either in crude extracts of uninfected E. coli or as a component of Qβ replicase) to the in vitro reaction specifically stimulates rRNA synthesis so that it accounts for 30% of the transcript. They also reported that ψr does not stimulate rRNA synthesis in the presence of guanosine tetraphosphate (a phosphorylated nucleoside accumulated in stringent cells starved for amino-acids)^6,7. Of the in vitro product, 10% is rRNA in the presence or absence of ψr.