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

Abstract 15N-Labeled Bacillus subtilis tRNATrp wild type and a series of mutants were hyperexpressed in Escherichia coli and purified for NMR studies with the use of two-dimensional nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) and three-dimensional NOESY-HSQC techniques. These made possible chemical shift assignments of imino protons and determination of the thermal stability of the tRNATrp molecules. 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 whose peaks were not clearly observed. Several base triplets found in the crystal structure of tRNA were observed in the present study as well. These studies also revealed two components of tRNATrp, which could not be separated by high pressure liquid chromatography, corresponding to s4U and U at position 8 of the tRNATrp, as indicated by two different sets of peaks for the TψC and D arms. The modification at position 8 altered the local 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 the s4U8 is more stable than the U8 component, thus providing evidence that the thiolation of U8 stabilizes the tertiary structure of tRNA.