Purification of Rhodobacter sphaeroides RNA Polymerase and Its Sigma Factors

Publisher Summary This chapter explores the rhodobacter sphaeroides that has a variety of metabolic lifestyles. In addition to generating energy by respiration or photosynthesis, this α-proteobacterium uses catabolic, anabolic, or energetic pathways of environmental, agricultural, or industrial interest. The chapter presents methods used to obtain R. sphaeroides core RNA polymerase and holoenzyme, along with the approaches used to purify active sigma factors or their cognate anti-sigma factors. It also outlines assays to monitor the ability of RNA polymerase to transcribe target genes, either alone or in the presence of specific transcription factors. Many of these methods are modifications of techniques used to monitor the activity of analogous proteins from other well-studied prokaryotes. The systems described are useful for analyzing the many transcriptional regulatory networks in R. sphaeroides and other bacteria.