Identification of three G.U base pairs in Bacillus subtilis ribosomal 5S RNA via 500-MHz proton homonuclear Overhauser enhancements.

Three distinct G.U base pairs in Bacillus subtilis 5S RNA have been identified via homonuclear Overhauser enhancements (NOE) of their low-field (9-15 ppm) proton Fourier transform nuclear magnetic resonances at 11.75 T. With these G.U resonances as starting points, short segments of NOE connectivity can be established. One G.U-G.C-G.C segment (most probably G4.C112-G5.C111-U6.G110) can definitely be assigned to the terminal helix. The existence of at least part of the terminal helical stem of the secondary structure of a Gram-positive bacterial 5S RNA has thus been established for the first time by direct experimental observation. Addition of Mg2+ produces almost no conformational changes in the terminal stem but results in major conformational changes elsewhere in the structure, as reflected by changes in the 1H 500-MHz low-field NMR spectrum. Assignment of the two remaining G.U base pairs will require further experiments (e.g., enzymatic-cleavage fragments). Finally, the implications of these results for analysis of RNA secondary structure are discussed.