Analysis of RNA Polymerase II Elongation In Vitro

This chapter describes methods useful for studying elongation by RNA polymerase II in vitro and provides illustrative examples of typical experiments. The protocol for preparation of nuclear extract is given. The quality of the results obtained from in vitro transcription reactions is directly related to the nuclear extract used. The most convenient way to examine elongation in vitro is to force synchronized initiation of polymerases and then follow the progress of the elongating polymerases with time. These conditions are met by the pulse–chase assay. Elongation complexes can be isolated using the immobilized template by exploiting the high stability of the RNA polymerase II ternary complex. Isolated elongation complexes are very useful in add-back assays to determine if a crude extract or purified protein has an effect on elongation. The chapter discusses capping assay and coupling of RNA processing events to transcription. Functional coupling is demonstrated using an immobilized template and an add-back assay. The add-back assay should be useful for examining other RNA processing events that may be functionally coupled to transcription, such as polyadenylation and splicing.