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. Biochemistry 1986 Jun 17;25(12):3673-82
Date
06/17/1986Pubmed ID
3087417DOI
10.1021/bi00360a030Scopus ID
2-s2.0-0023051831 (requires institutional sign-in at Scopus site) 15 CitationsAbstract
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.
Author List
Li SJ, Marshall AGMESH terms used to index this publication - Major topics in bold
Base CompositionBase Sequence
Endoribonucleases
Magnetic Resonance Spectroscopy
Nucleic Acid Conformation
Oligoribonucleotides
Plants
RNA, Ribosomal
Ribonuclease T1
Ribosomes
Triticum
