Identification and characterization of modification enzymes by biochemical analysis of the proteome

The use of proteomic libraries designed to express the complete set of proteins from an organism has resulted in the identification of many RNA modification enzymes whose function was previously unknown. Here we describe a generalized procedure for the biochemical analysis of a yeast proteomic library for identification of nucleic acid-modifying enzymes, by use of the yeast MORF (Moveable Open Reading Frame) library (Gelperin et al., 2005) as the source of protein activity, and the known yeast tRNA methyltransferase Trm4 as a test case. The procedures outlined in this chapter can be applied to any proteomic expression library from any organism, many of which will become increasingly available as the number of sequenced genomes increases and as genomic cloning techniques improve.