Transcription in vitro of DNA in isolated bacterial nucleoids

Abstract

The in vitro transcription of DNA in isolated nucleoids from Escherichia coli was investigated. The purpose of the study was to determine the effects on DNA transcription of using as template a condensed bacterial chromosome. To compare the template capacity of the DNA in the condensed chromosome with that of the completely unfolded chromosome, very high RNA polymerase/DNA ratios were used during transcription so that the amount of DNA was limiting. The rate of total RNA synthesis was greater on the condensed chromosome. The RNA chain elongation rate and the average size of the RNA transcripts were similar on the condensed and unfolded templates; however, RNA chain initiation rates were greater on the condensed chromosome. The results show that the maximum number of RNA polymerase molecules which can simultaneously synthesize RNA is greater on the condensed chromosome. A preferential synthesis of ribosomal RNA was observed on both the condensed and unfolded chromosomes. Although the absolute rate of rRNA synthesis was greater on the condensed template, the rate of rRNA synthesis relative to the rate of total RNA synthesis was similar on both templates. The results suggest that the genes specifying rRNA do not differ significantly in their availability for transcription relative to the remainder of the genome in folded and unfolded chromosomes. Moderate concentrations of spermidine stabilized the condensed state of the folded chromosome during transcription and the average size of the isolated nucleoids, as estimated from sedimentation studies and direct microscopic visualization, remained unchanged during transcription.