Isolation and functional analysis of RNA polymerase II elongation complexes.

摘要

The elongation phase of transcription by RNA polymerase II (RNAP II) is tightly controlled by a large number of transcription elongation factors. Here we describe experimental approaches for the isolation of RNAPII elongation complexes in vitro and the use of these complexes in the examination of the function of a variety of factors. The methods start with formation of elongation complexes on DNA templates immobilized to paramagnetic beads. Elongation is halted by removing the nucleotides and the ternary elongation complexes are then stripped of factors by a high salt wash. The effect of any factor or mixture of factors on elongation is determined by adding the factor(s) along with nucleotides and observing the change in the pattern of RNAs generated. Association of a factor with elongation complexes can be examined using an elongation complex-electrophoretic mobility shift assay (EC-EMSA) in which elongation complexes that have been liberated from the beads are analyzed on a native gel. Besides being used to dissect the mechanisms of elongation control, these experimental systems are useful for analyzing the function of termination factors and mRNA processing factors. Together these experimental systems permit detailed characterization of the molecular mechanisms of elongation, termination, and mRNA processing factors by providing information concerning both physical interactions with and functional consequences of the factors on RNAPII elongation complexes.