Localization and functional analysis of the regulated promoter from the Streptomyces glaucescens mel operon

Abstract

The transcription initiation site of the mel operon from Streptomyces glaucescens, determined by S1 mapping and primer elongation experiments, lies 32 to 34 bp upstream of the translation initiation codon of the first open reading frame. A total of 172 to 219 bp upstream of the transcription start point are necessary for a fully active and regulated mel promoter. Deletion analysis, gel retardation assays and DNAse I footprint experiments facilitated division of the promoter into three functional domains, which include the RNA polymerase recognition site up to nucleotides -33 to -42, the binding region of a protein of assumed regulatory function between nucleotides -65 and -93, and an upstream activator site, located between positions -158 and -219.

Cloning, sequencing, and expression of the gene encoding methylmalonyl-coenzyme A mutase from Streptomyces cinnamonensis

“…RNA was isolated from 2-to 6-day-old cultures by the method of Hopwood et al (15). DNA fragments were end-labelled by the method of Sambrook et al (39); hybridization, S1 digestion, and analysis were performed as detailed by Geistlich et al (12).…”