Bacillus subtilis tRNA^Trp wild-type and a series of mutants hyper-expressed in E. coli were studied by using multinuclear and multidimensional NMR techniques. Most of the imino protons in the helical regions and of the tertiary base pairs were assigned. Several base triplets found in the crystal structure of tRNA were observed in the present study as well. Two forms of tRNA^Trp, bearing s⁴U8 or U8, were observed. The modification at position 8 alters the local conformation of the core region of the tRNA and makes it more stable than the unmodified species. ^Trp...[ Read more ]

Bacillus subtilis tRNA^Trp wild-type and a series of mutants hyper-expressed in E. coli were studied by using multinuclear and multidimensional NMR techniques. Most of the imino protons in the helical regions and of the tertiary base pairs were assigned. Several base triplets found in the crystal structure of tRNA were observed in the present study as well. Two forms of tRNA^Trp, bearing s⁴U8 or U8, were observed. The modification at position 8 alters the local conformation of the core region of the tRNA and makes it more stable than the unmodified species.

Mutation of U11A24, one of the major identity elements of tRNA^Trp, to C11G24 causes local conformational changes in the core region. Magnesium titration experiments showed that there is at least one strong binding site near position 8. The binding affinity of this site in the C11G24 mutant is reduced by about two folds compared to that in wild type. The results show that the U11A24 base pair is necessary for the stabilization of the special conformation in the core region, which may be important for the recognition of tRNA^Trp by its cognate aminoacyl-tRNA synthetase.

The hydrogen bond network in tRNA^Trp was characterized by J_NN HNN-COSY experiment, by which the scalar couplings across hydrogen bonds between the donor and acceptor nitrogen atoms, ^2hJ_NN, can be derived. For tRNA^Trp, all the secondary and tertiary base pairs, as well as a base triple, with hydrogen bonds between proton and nitrogen, were observed to give cross peaks in the spectrum. This is the direct evidence for the existence of these hydrogen bonds. A new method was proposed to measure another coupling constant across hydrogen bond, ^lhJ_HN, of large molecules.

To delineate the effect of the discriminator base on aminoacylation, two RNA microhelices, derived from the acceptor stem of tRNA^Trp wild type and A73 mutant, were constructed and studied by NMR spectroscopy. The results show that both molecules possess A-form like conformations in the stem region and the 3'-terminals. No significant conformational difference was found between them. It is then suggested that some group in the discriminator base may play an important role in the recognition of tRNA^Trp by TrpRS.