Simple Fluorescence Assays Probing Conformational Changes of Escherichia coli RNA Polymerase During Transcription Initiation

Publisher Summary This chapter describes a simple temperature-dependent fluorescence assay of the gradual conformation changes of RNAP in these intermediates. Assays involve a wide range of temperature from 4o to 37oC in order to probe as many intermediate states as possible. Simple fluorescence assays to monitor the conformational changes of RNAP during the formation of an open complex at trp promoter are described. The assays are more informative if RNAP with different σ subunits along with the different promoter mutants with known biochemical phenotypes. Another interesting application studies the effect of repressor and activator molecules on RNAP–promoter combinations. It is expected that the fluorescence properties of either the active site or the whole RNAP alter in response to the repressor and activator molecules. Finally, as fluorescence signals are indirect measurements of conformational changes, it is important to use these techniques on biochemically characterized RNAP–promoter complexes. If these were to be applied to a new system, it is advisable to perform parallel biochemical assays such as DNA footprinting to confirm the existence of intermediate states.