Production, purification, and assay of recombinant proteins for in vitro biochemical analyses of the plant polyadenylation complex

Abstract

In the post-genomic era where gene sequences are available for many organisms, attention has shifted from DNA to the workhorses of the cell—RNA and protein. A number of proteins, as recent studies indicate, seem to possess RNA-binding and RNA cleavage activities. In order to understand the events that comprise RNA processing such as splicing, 3′ end processing, and even RNA turnover, well established methods are necessary. Bacterial recombinant proteins afford an invaluable opportunity to produce proteins in an economical and reproducible fashion in order to study these activities. This chapter describes various experimental protocols to begin the elucidation of the many events that surround RNA processing at the 3′ end of a transcript.