This is a second revision of a proposal to measure certain structural features of tRNA in solution. Using a technique developed in his own laboratory, Dr. Hagerman can measure the angle between major stems in tRNA (and other RNAs) by comparing rotational diffusion times of a tRNA heteroduplex and a linear RNA of the same length. In particular, it is proposed to measure the """"""""L-angle"""""""" in tRNAs, the angle between the anti-codon and acceptor stems. For this work, the heteroduplex will be artificially extended by placing about 70 extra base pairs on the anti-codon and acceptor stems. Some mitochondrial tRNAs, for features suchas the D-loop or Tpsi C-loop appear to be missing. Recent modeling work suggests that these atypical tRNAs also possess atypical tRNA structure in 3 dimensions. Thus, the """"""""L-angle"""""""" for several of these atypical mitochondrial tRNAs will be measured to determine whether or not these models are consistent with data. The methods proposed here yield information not available from crystallographic or other solution studies. They can measure not only interstem angles, but also the range and flexibility of those angles. Thus the """"""""L-angle"""""""" and its range will be measured under different ionic conditions and with different tertiary interactions. Studying these gross structural changes when bases are altered is relevant to certain neuromuscular diseases caused by mutations in mitochondrial tRNAs.
