Identification and assignment of base pairs in the "tuned helix" of intact and ribonuclease T1 cleavage fragments of wheat germ ribosomal 5S RNA via 500-MHz proton homonuclear Overhauser enhancements.

Abstract

Wheat germ has been chosen as a representative eukaryote for study of ribosomal 5S RNA secondary structure. Proton homonuclear Overhauser enhancements (NOE's) at 500 MHz for the hydrogen-bonded base-pair protons in the 10-15 ppm region are used to establish the identity (A X U, G X C, or G X U) and base-pair sequence (e.g., G X C-A X U-C X G) within a given helical segment. Assignment of that segment to particular base pairs in the secondary structure is based upon NOE's conducted at different temperatures (to determine which signals "melt" together), variation of salt conditions (to produce differential chemical shifts in order to better distinguish components of an unresolved spectral envelope), and isolation and purification of RNase T1 cleavage fragments (in order to reduce the spectrum to just a few base pairs). The NOE patterns for the RNase T1 fragments are the same as in the intact 5S RNA, supporting the assumption that structural features of this region in the intact 5S RNA are preserved in the fragment. Chemical shifts predicted from ring current induced effects for a proposed base-pair sequence are then compared to experimental chemical shifts. By these methods, a portion of the "tuned helix" segment (namely, the base-pair sequence C18G60-A19U59-C20G58) is demonstrated spectroscopically for the first time in any 5S RNA. The tuned helix and common arm segments are less stable than the rest of the molecule. Variation of sodium and magnesium levels reveals multiple configurations of the wheat germ 5S RNA in solution.