Synthesis of a hetero subunit RNA modification enzyme by the wheat germ cell-free translation system

Abstract

Cell-free translation systems are a powerful tool for the production of many kinds of proteins. However, there are some barriers to improve the system in order to make it a more convenient approach. These include the fact that the production of proteins made up of hetero subunits is difficult. In this chapter, we describe the synthesis of yeast tRNA (m(7)G46) methyltransferase as a model protein. This enzyme catalyzes transfer of a methyl group from S-adenosyl-L-methionine to guanine at position 46 in tRNA and generates N(7)-methylguanine. Yeast tRNA (m(7)G46) methyltransferase is composed of two protein subunits, Trm8 and Trm82. To obtain the active Trm8-Trm82 complex, co-translation of both subunits is necessary. Preparation of mRNAs, in vitro synthesis and purification of the complex are explained in this chapter.