Fluorophore labeling to monitor tRNA dynamics

Transfer RNA (tRNA) molecules mediate translation of the nucleic acid genetic code into the amino acid building blocks of proteins, thus ensuring the survivability of cells. The dynamic properties of tRNA molecules are crucial to their functions in both activity and specificity. This chapter summarizes two methods that have been recently developed or improved upon previous protocols to introduce fluorophores to site-specific positions in tRNA. One method enables incorporation of fluorophores carrying a primary amine (such as proflavin or rhodamine) to dihydrouridine (D) residues in the tRNA tertiary core, and a second method enables incorporation of pyrroloC and 2-aminopurine to positions 75 and 76, respectively, of the CCA sequence at the 3' end. These site-specific fluorophore labeling methods utilize tRNA transcripts as the substrates to provide the versatility with both wild-type and mutant sequences for examining their motions in space and time during the process of decoding genetic information.