Kinetics of Multisubunit RNA Polymerases: Experimental Methods and Data Analysis

Abstract

Publisher Summary This chapter summarizes the transient-state kinetic methods and subsequent data analysis that can be used to determine the kinetic mechanisms of transcription elongation. To observe single or multiple nucleotide incorporation during the elongation phase of transcription, it is essential to form stalled complexes. Such complexes permit the synchronization of RNA polymerase (RNAP) on the DNA template. Stalled elongation complexes can be formed using nucleotide starvation. The stalled elongation complexes must be purified before single nucleotide incorporation experiments can be conducted. By using a biotinylated DNA template bound to streptavidin-coated magnetic beads, the complexes can be purified by placing the reaction tube next to a magnetic eppendorf tube rack and washing away the NTPs. After washing, the complexes can be used in kinetic experiments to observe single or multiple nucleotide addition. Alternatively, the RNAP can be “walked” down the template to the next position or subsequent positions to start at a different stall site. This procedure simply involves incubating the complexes with the next NTP(s) and purifying the complexes again.