26] Mapping the structure of specific protein-transfer RNA complexes by a tritium labeling method

Abstract

Publisher Summary The chapter discusses the technique of mapping the structure of specific protein-transfer RNA complexes by a tritium labeling method. Tritium labeling of purines is an ideal and gentle way to map the structure of protein-nucleic acid complexes. This is done by determining the labeling rates of the various bases in a nucleic acid in the presence and in the absence of the bound protein. Tritium is an atomic-size probe, therefore, it provides a level of structural discrimination greater than that achievable with a more bulky reagent. The general features of the tritium labeling reaction as applied to protein-nucleic acid complexes are discussed. A specific application is illustrated with the complex between an aminoacyltRNA synthetase and its cognate tRNA. For exploring protein-nucleic acid complexes, the simplest preliminary experiment is to check the effect of a bound protein on the labeling rate of a mononucleotide. A straightforward experiment is to examine the effect of an aminoacyl tRNA synthetase on the labeling rate of the substrate ATP. The main objective is to determine the tritium labeling rates of free and bound nucleotide units distributed throughout a nucleic acid structure. In the chapter, an illustration is given for protein-tRNA complexes; however, the general procedures and ideas are applicable to virtually any protein-nucleic acid system.