Use of exhaustive nucleic acid hybridization for determining the amount and size distribution of newly synthesized bacterial message RNA.

Abstract

An RNA-DNA hybridization method is described for determining the fraction of a radioisotopically labeled RNA preparation isolated from bacteria that is message RNA. Experiments are performed under conditions where over 95% of the input RNA is converted to an RNA-DNA complex. Competitive hybridization methods are described that partition the RNA-DNA hybrids into ribosomal RNA-DNA and nonribosomal RNA-DNA hybrids. Evidence is presented that the competition is specific. Message RNA radioactivity was assumed to be RNA radioactivity not competed by ribosomal RNA and transfer RNA. The fraction of RNA made at a given instant under balanced growth conditions (30 C, in minimal medium), that is, message RNA, was determined to be 65%. The method was also used to measure the size distribution of newly synthesized mRNA in Escherichia coli. That size was found to average 3–8 × 105 daltons.