NMR ANALYSIS OF TRANSFER-RNA ACCEPTOR STEM MICROHELICES - DISCRIMINATOR BASE CHANGE AFFECTS TRANSFER-RNA CONFORMATION AT THE 3' END

An important step in initiation of protein synthesis in Escherichia co li is the specific formylation of the initiator methionyl-tRNA (Met-tR NA) by Met-tRNA transformylase. The determinants for formylation are c lustered mostly in the acceptor stem of the initiator tRNA. Here we us e NMR spectroscopy to characterize the conformation of two RNA microhe lices, which correspond to the acceptor stem of mutants off. coli init iator tRNA and which differ only at the position corresponding to the ''discriminator base'' in tRNAs. One of the mutant tRNAs is an extreme ly poor substrate for Met-tRNA transformylase, whereas the other one i s a muck better substrate. We show that one microhelix forms a structu re in which its 3'-(A) under bar CCA sequence extends the stacking of the acceptor stem. The other microhelix forms a structure in which its 3'-(U) under bar CCA sequence folds back such that the 3' terminal A2 2 is in close proximity to G1. These results highlight the importance of the discriminator base in determining tRNA conformation at the 3' e nd. They also suggest a correlation between tRNA structure at the 3' e nd and its recognition by Met-tRNA transformylase.