Rho-dependent transcriptional polarity in the ilvGMEDA operon of wild-type Escherichia coli K12.

It has been generally accepted that transcriptional polarity in prokaryotic systems is due to an uncoupling of translation and transcription which unmasks latent rho-dependent termination sites in a polycistronic messenger RNA. In this report, we identify and characterize rho-dependent termination sites responsible for transcriptional polarity in the ilvGMEDA operon of wild-type Escherichia coli K12. The ilvG gene in the wild-type E. coli K12 ilvGMEDA operon contains a frameshift site which results in termination of translation in the middle of the gene. Mutations have been characterized which restore the reading frame of this gene. In addition to allowing full-length expression of the ilvG product, these mutations cause a 3-4-fold elevation in the expression of the operon distal genes. This transcriptional polarity effect on operon distal genes also has been shown to be relieved by rho suppressor mutations. We have used in vitro transcription experiments to identify rho-dependent transcriptional termination sites downstream of the frameshift site in the ilvG gene. Three tandem rho-dependent sites have been located in the ilv'GM' gene region using transcription reactions containing linear or supercoiled plasmid DNA templates. Accumulatively, these rho-dependent termination sites account for about 80% in vitro transcription termination, which is in agreement with the in vivo measurements of transcriptional polarity on operon distal gene expression. These transcriptional experiments provide in vitro confirmation for the latent rho-dependent termination site model of transcriptional polarity.