NMR studies of Bacillus subtilis tRNA(Trp) hyperexpressed in Escherichia coli - Assignment of imino proton signals and determination of thermal stability

N-15-Labeled Bacillus subtilis tRNA(Trp) wild type and a series of mutants were hyperexpressed in Escherichia coli and purified for NMR studies with t he use of two-dimensional nuclear Overhauser effect spectroscopy (NOESY) an d heteronuclear single quantum correlation (HSQC) and three-dimensional NOE SY-HSQC techniques, These made possible chemical shift assignments of imino protons and determination of the thermal stability of the tRNATrp molecule s. Almost all of the imino protons in the helical regions and the tertiary base pairs were assigned, except three imino protons of the AU base pairs w hose peaks were not clearly observed. Several base triplets found in the cr ystal structure of tRNA were observed in the present study as well. These s tudies also revealed two components of tRNA(Trp), which could not be separa ted by high pressure liquid chromatography, corresponding to s(4)U and U at position 8 of the tRNA(Trp), as indicated by two different, sets of peaks for the T psi C and D arms. The modification at position 8 altered the loca l conformation of the core region of the tRNA Thermal unfolding experiments showed that the unfolding process is cooperative in the presence of a high concentration of magnesium ions and that the component corresponding to th e s(4)U8 is more stable than the U8 component, thus providing evidence that the thiolation of U8 stabilizes the tertiary structure of tRNA.