A semi-quantitative pull-down assay to study tRNA substrate specificity of modification enzymes.

ABSTRACT

A tRNA interacts with numerous proteins throughout its biogenesis and during translation, and a significant portion of these interacting proteins are involved in post-transcriptional modifications. While some of the modifying enzymes use relatively simple recognition elements for substrate recognition, many enzymes selectively modify a specific subset of tRNA species without obvious recognition rules. In this chapter we describe a semi-quantitative pull-down assay to study tRNA substrate specificity of modification enzymes, by using the yeast Saccharomyces cerevisiae m3C32 methyltransferase Trm140 as an example. We also discuss some overall considerations for a successful pull-down experiment, with a focus on practical applications of the dissociation constant KD between the protein and the tRNA and the off-rate.