Effect of G-1 on Histidine tRNA Microhelix Conformation

Seetharaman, M.; Williams, C.; Cramer, C. J.; Musier-Forsyth, K.
Nucl. Acids Res. 2003, 31, 7311.

Histidine tRNAs (tRNA^His) are unique in that they possess an extra 5'-base (G-1), not found in other tRNAs. Deletion of G-1 results in at least a 250-fold reduction in the rate of histidine charging in vitro. To better understand the role of the G-1 nucleotide in defining the structure of tRNA^His, and to correlate structure to cognate amino acid charging, NMR and molecular dynamics (MD) studies were performed on the wild-type and a DG-1 mutant Escherichia coli histidine tRNA acceptor stem microhelix. Using NMR-derived distance restraints, global structural characteristics are described and interpreted to rationalize experimental observations with respect to aminoacylation activity. The quality of the NMR-derived solution conformations of the wild-type and DG-1 histidine microhelices is assessed using a variety of MD-based computational protocols. Most of the duplex regions of the acceptor stem and the UUCG tetraloop are well defined and effectively superimposable for the wild-type and DG-1 mutant microhelix^His. Differences, however, are observed at the end of the helix and in the single-stranded CCCA-3' tail. The wild-type microhelixHis structure is more well-defined than the mutant and folds into a "stacked fold-back" conformation. In contrast, we observe fraying of the first two base pairs and looping back of the single-stranded region in the DG-1 mutant resulting in a much less well-defined conformation. Thus, the role of the extra G-1 base of the unique G-1:C73 base pair in tRNA^His may be to prevent end-fraying and stabilize the stacked fold-back conformation of the CCCA-3' region.

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