Structural sites of RNA synthesis in Escherichia coli

Abstract

This work describes an attempt to isolate and study naturally occurring mRNA-DNA complexes from bacteria. When normal or T4-infected Escherichia coli cells were lysed gently in the cold using a lysozyme-EDTA treatment, all the bacterial, or bacterial and phage, DNA was sedimentable with the membranous fraction. 85% or more of the rapidly labeled RNA, and from 30 to 45% of the metabolically stable RNA was found in association with this membrane-bound DNA. The mRNA-membrane DNA complex was shown to be in a dynamic state by kinetic studies, using cumulative labeling or chase experiments. It was thus shown that the sedimentable mRNA fraction was the precursor for mRNA chains which appeared later in the cytoplasmic fraction. Sedimentation analyses of the RNA recovered from the membrane DNA fraction revealed that it was made of chains bound to 30 or 70 s ribosomes, or multiribosomal complexes. A partial purification of the crude membrane-DNA fraction based on a sucrose-gradient centrifugation after a Mg-sarkosyl treatment is described. A DNA-enriched fraction sedimenting as a rather homogeneous component was obtained. Most of the newly synthesized T4-specific RNA was found in a metabolically renewable association with this purified nuclear membrane complex. These results are discussed in terms of their significance for studying mRNA transcription and release in a structurally integrated system.