Karin M. Musier-Forsyth, Principal Investigator

Importance of Discriminator Base Stacking Interactions: Molecular Dynamic Analysis of A73 Microhelix^Ala and Microhelix^His Variants

Transfer of alanine from Escherichia coli alanyl-transfer ribonucleic acid (tRNA) synthetase (AlaRS) to RNA minihelices that mimic the amino acid acceptor stem of tRNAAla was shown, by analysis of variant minihelix aminoacylation activities, to involve a transition state sensitive to changes in the “discriminator” base at position 73. Solution nuclear magnetic resonance (NMR) indicated that this single-stranded nucleotide is predominantly stacked onto G1 of the first base pair of the alanine acceptor stem helix. These researchers observed the activity of a new variant with the adenine at position 73 substituted by its non-polar isostere 4-methylindole (M). Despite lacking N7, this analog is well tolerated by AlaRS. Molecular dynamics (MD) simulations show that the M substitution improves position 73 base stacking over G1, as measured by a stacking lifetime analysis. Additional MD simulations of wild-type microhelixAla and six variants reveal a positive correlation between N73 base stacking propensity over G1 and aminoacylation activity. For the two DN7 variants simulated, the researchers found that the propensity to stack over G1 was similar to the analogous variants that contain N7, leading to the conclusion that the decrease in amino-acylation efficiency observed upon deletion of N7 is likely due to loss of a direct stabilizing interaction with the synthetase.

These researchers also performed work in conformational analysis of histidine tRNA acceptor stem models. Histidine tRNA is unique in that its acceptor stem has only a single strand of three nucleotides rather than the four nucleotides common to most tRNAs. This is due to the fact that the discriminator base (C73) is paired with a G-1. Conformations of several tRNA acceptor stems have previously been studied to determine the role of the tRNA conformation in recognition by the cognate synthetase. However, the effect of a C in the discriminator position has never been studied and the conformation of the unique tRNAHis acceptor stem sequence is unknown. The goal of this project is to determine the conformation of the acceptor stem model microhelixHis, both wild-type (with a G at position -1) and without the G-1. To date, the RNA samples have been prepared and solution NMR experiments have been completed. Constraint files were generated to model the acceptor stems using the program amber. Molecular dynamics simulations were carried out with the NMR generated distance constraints, solvated with explicit water.

Research Group and Collaborator

Penny Beuning, Graduate Student Researcher
Christopher J. Cramer, Faculty Collaborator
Maria C. Nagan, Graduate Student Researcher
Caroline Williams, Research Associate